Treatment of brain derived neurotrophic factor (bdnf) related diseases by inhibition of natural antisense transcript to bdnf

ABSTRACT

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of Brain derived neurotrophic factor (BDNF), in particular, by targeting natural antisense polynucleotides of Brain derived neurotrophic factor (BDNF). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of BDNF.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Prov. Pat. App. Ser. No.61/611,225, filed on Mar. 15, 2012; and U.S. Prov. Pat. App. Ser. No.61/614,664, filed on Mar. 23, 2012, both of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

Embodiments of the invention comprise oligonucleotides modulatingexpression and/or function of BDNF and associated molecules.

BACKGROUND

DNA-RNA and RNA-RNA hybridization are important to many aspects ofnucleic acid function including DNA replication, transcription, andtranslation. Hybridization is also central to a variety of technologiesthat either detect a particular nucleic acid or alter its expression.Antisense nucleotides, for example, disrupt gene expression byhybridizing to target RNA, thereby interfering with RNA splicing,transcription, translation, and replication. Antisense DNA has the addedfeature that DNA-RNA hybrids serve as a substrate for digestion byribonuclease H, an activity that is present in most cell types.Antisense molecules can be delivered into cells, as is the case foroligodeoxynucleotides (ODNs), or they can be expressed from endogenousgenes as RNA molecules. The FDA recently approved an antisense drug,VITRAVENE™ (for treatment of cytomegalovirus retinitis), reflecting thatantisense has therapeutic utility.

WO 2010/093904 and its US counterpart US/2011/0319475 disclose BDNF as atarget for modulation using oligonucleotides recited therein. There is aneed for continued development with respect to natural antisense targetsand newly developed oligonucleotides that complement such targets andmodulate BDNF protein expression to potentially treat or be used inresearch associated with treating BDNF related diseases and conditions.

SUMMARY

This Summary is provided to present a summary of the invention tobriefly indicate the nature and substance of the invention. It issubmitted with the understanding that it will not be used to interpretor limit the scope or meaning of the claims.

In one embodiment, the invention provides methods for inhibiting theaction of a natural antisense transcript by using antisenseoligonucleotide(s) targeted to any region of the natural antisensetranscript resulting in up-regulation of the corresponding BDNF sensegene in mammalian organisms. It is also contemplated herein thatinhibition of the natural antisense transcripts recited herein can beachieved by siRNA, ribozymes and small molecules, which are consideredto be within the scope of the present invention.

One embodiment provides a method of modulating function and/orexpression of a BDNF polynucleotide in biological systems, including,but not limited to, patient cells or tissues in vivo or in vitrocomprising contacting said biological system or said cells or tissueswith an antisense oligonucleotide of about 5 to about 30 nucleotides inlength wherein said oligonucleotide has at least 50% sequence identityto a reverse complement of a polynucleotide comprising 5 to 30consecutive nucleotides within nucleotides 1 to 1279 of SEQ ID NO: 3 or1 to 1478 of SEQ ID NO: 4 or 1 to 1437 of SEQ ID NO: 5 or 1 to 2322 ofSEQ ID NO: 6 or 1 to 2036 of SEQ ID NO: 7 or 1 to 2364 of SEQ ID NO: 8or 1 to 3136 of SEQ ID NO: 9 or 1 to 906 of SEQ ID NO: 10 or 1 to 992 ofSEQ ID NO: 11 thereby modulating function and/or expression of the BDNFpolynucleotide in said biological system including said patient cells ortissues in vivo or in vitro, with the proviso that the oligonucleotideshaving SEQ ID NOS 50-55 are excluded.

In an embodiment, an oligonucleotide recited above targets a naturalantisense sequence of BDNF polynucleotides present in a biologicalsystem, for example, nucleotides set forth in SEQ ID NOS: 3 to 11, andany variants, alleles, homologs, mutants, derivatives, fragments andcomplementary sequences thereto. Examples of such antisenseoligonucleotides are set forth as SEQ ID NOS: 12 to 49.

In another embodiment, the invention comprises a method of modulatingthe function or expression of a BDNF polynucleotide in a biologicalsystem comprising contacting said biological system with at least oneantisense oligonucleotide that targets a natural antisense transcript ofthe BDNF polynucleotide comprising 5 to 30 consecutive nucleotideswithin nucleotides 1 to 1279 of SEQ ID NO: 3 or 1 to 1478 of SEQ ID NO:4 or 1 to 1437 of SEQ ID NO: 5 or 1 to 2322 of SEQ ID NO: 6 or 1 to 2036of SEQ ID NO: 7 or 1 to 2364 of SEQ ID NO: 8 or 1 to 3136 of SEQ ID NO:9 or 1 to 906 of SEQ ID NO: 10 or 1 to 992 of SEQ ID NO: 11 therebymodulating the function and/or expression of the BDNF polynucleotide insaid biological system.

In another embodiment, the invention comprises a method of modulatingthe function or expression of a BDNF polynucleotide in a biologicalsystem comprising contacting said biological system with at least oneantisense oligonucleotide that targets a region of a natural antisensetranscript of the BDNF polynucleotide comprising 5 to 30 consecutivenucleotides within nucleotides 1 to 1279 of SEQ ID NO: 3 or 1 to 1478 ofSEQ ID NO: 4 or 1 to 1437 of SEQ ID NO: 5 or 1 to 2322 of SEQ ID NO: 6or 1 to 2036 of SEQ ID NO: 7 or 1 to 2364 of SEQ ID NO: 8 or 1 to 3136of SEQ ID NO: 9 or 1 to 906 of SEQ ID NO: 10 or 1 to 992 of SEQ ID NO:11 thereby modulating the function and/or expression of the BDNFpolynucleotide in said biological system.

In an embodiment, the invention comprises a method of increasing thefunction and/or expression of a BDNF polynucleotide having SEQ ID NO. 1and 2 in a biological system comprising contacting said biologicalsystem with at least one antisense oligonucleotide that targets anatural antisense transcript of said BDNF polynucleotide comprising 5 to30 consecutive nucleotides within nucleotides 1 to 1279 of SEQ ID NO: 3or 1 to 1478 of SEQ ID NO: 4 or 1 to 1437 of SEQ ID NO: 5 or 1 to 2322of SEQ ID NO: 6 or 1 to 2036 of SEQ ID NO: 7 or 1 to 2364 of SEQ ID NO:8 or 1 to 3136 of SEQ ID NO: 9 or 1 to 906 of SEQ ID NO: 10 or 1 to 992of SEQ ID NO: 11 thereby increasing the function and/or expression ofsaid BDNF polynucleotide or expression product thereof.

In another embodiment, the invention comprises a method of method ofincreasing the function and/or expression of a BDNF polynucleotidehaving SEQ ID NO. 1 and 2 in a biological system comprising contactingsaid biological system with at least one antisense oligonucleotide thattargets a natural antisense transcript of said BDNF polynucleotidethereby increasing the function and/or expression of said BDNFpolynucleotide or expression product thereof wherein the naturalantisense transcripts are selected from SEQ ID NOS. 3 to 11.

In another embodiment, the invention comprises a method of method ofincreasing the function and/or expression of a BDNF polynucleotidehaving SEQ ID NO. 1 and 2 in a biological system comprising contactingsaid biological system with at least one antisense oligonucleotide thattargets a natural antisense transcript of said BDNF polynucleotidethereby increasing the function and/or expression of said BDNFpolynucleotide or expression product thereof wherein the naturalantisense transcripts are selected from SEQ ID NOS. 3 to 11 and whereinthe antisense oligonucleotides are selected from at least one of SEQ IDNOS. 12 to 49.

In an embodiment, a composition comprises one or more antisenseoligonucleotides which bind to sense and/or antisense BDNFpolynucleotides.

In an embodiment, the oligonucleotides comprise one or more modified orsubstituted nucleotides.

In an embodiment, the oligonucleotides comprise one or more modifiedbonds.

In yet another embodiment, the modified nucleotides comprise modifiedbases comprising phosphorothioate, methylphosphonate, peptide nucleicacids, 2′-O-methyl, fluoro- or carbon, methylene or other locked nucleicacid (LNA) molecules. Preferably, the modified nucleotides are lockednucleic acid molecules, including α-L-LNA.

In an embodiment, the oligonucleotides are administered to a patientsubcutaneously, intramuscularly, intravenously or intraperitoneally.

In an embodiment, the oligonucleotides are administered in apharmaceutical composition. A treatment regimen comprises administeringthe antisense compounds at least once to patient; however, thistreatment can be modified to include multiple doses over a period oftime. The treatment can be combined with one or more other types oftherapies.

In an embodiment, the oligonucleotides are encapsulated in a liposome orattached to a carrier molecule (e.g. cholesterol, TAT peptide).

In an embodiment, the present invention comprises the use of SEQ ID NOS50-55 as oligonucleotides targeting the natural antisense transcripts(NATs) to modulate the expression of a BDNF polynucleotide wherein saidNATs are selected from the group consisting of SEQ ID NOS. 3 to 11. Inanother embodiment, the present invention comprises the use of SEQ IDNOS 50-55 as oligonucleotides targeting the natural antisensetranscripts (NATs) to modulate the expression of a BDNF polynucleotidewherein said NATs are selected from the group consisting of SEQ ID NOS.3, 4, 5, 7, 8, 9, 10 and 11.

Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-e show antisense-mediated regulation of sense mRNA andprotein. FIG. 1 a shows that after transfection of several human andmouse cell lines with three siRNA oligonucleotides, targeted tonon-overlapping regions of the BDNF-AS transcript, knockdown andupregulation of BDNF transcript occurred. FIG. 1 b shows time coursestudy data after administration of BDNF-AS-targeted siRNA in theendogenous expression of both BDNF and BDNF-AS transcripts. The datashows that over the course of time BDNF-AS is downregulated and thenBDNF expression is upregulated and is reversible. FIG. 1 c shows thatBDNF protein, measured by ELISA, was significantly increased with twosiRNAs targeting BDNF-AS transcript, but not with scrambled siRNAs or acontrol nontargeting siRNA. FIG. 1 d shows protein levels of BDNFfollowing administration of various siRNAs using ELISA and/or westernblotting. FIG. 1 e shows the fold change percentage of BDNF compared tomock control versus increasing concentrations of oligonucleotide (10⁻¹²to 10⁻⁶ M).

FIG. 2 shows Bdnf upregulation increases neuronal outgrowth.

FIG. 3 shows Bdnf-AS regulates Bdnf mRNA and protein in vivo.

FIG. 4 shows Blocking of Bdnf-AS, in vivo, causes an increase inneuronal survival and proliferation.

FIG. 5 shows BDNF-AS knockdown leads to BDNF mRNA upregulation.

FIG. 6 shows Posttranscriptional regulation of Bdnf expression.

FIG. 7 shows Inhibition of the human BDNF-AS transcript byhBDNFAntagoNAT.

FIG. 8 shows Inhibition of the mouse Bdnf-AS transcript in N2a cells, byAntagoNATs.

FIG. 9 shows BDNF-AS knockdown neither changes the level of TrkB norBDNF neighboring genes (Let7C and KIF18A) in both directions: LIN7C andKIF18A are genes located 3′ downstream and 5′ upstream of BDNF,respectively.

Sequence Listing Description: SEQ ID NO: 1: Homo sapiens Brain derivedneurotrophic factor (BDNF), transcript variant 3, mRNA. (NCBI AccessionNo.: NM_(—)170735); SEQ ID NO: 2: Mus musculus brain derivedneurotrophic factor (Bdnf), transcript variant 1, mRNA (NCBI AccessionNo.: NM_(—)007540); SEQ ID NO: 3: Natural BDNF antisense sequence(transcript variant BT1A; NR_(—)033313.1); SEQ ID NO: 4: Natural BDNFantisense sequence (transcript variant BT2A; NR_(—)033314.1); SEQ ID NO:5: Natural BDNF antisense sequence (transcript variant BT1B;NR_(—)033315.1); SEQ ID NO: 6: Natural BDNF antisense sequence(transcript variant BT2B; NR_(—)002832.2); SEQ ID NO: 7: Natural BDNFantisense sequence (transcript variant BT1C; NR_(—)033312.1); SEQ ID NO:8: Natural BDNF antisense sequence (BDNF-AS variant); SEQ ID NO: 9:Natural BDNF antisense sequence; SEQ ID NO: 10: Mouse natural BDNFantisense sequence (Mouse BDNF-AS variant 1); SEQ ID NO: 11: Mousenatural BDNF antisense sequence (Mouse BDNF-AS variant 2); SEQ ID NOs:12 to 55: Antisense oligonucleotides; SEQ ID NO: 56 to 59: Reversecomplement of the antisense oligonucleotides 12 to 15 respectively; SEQID NO: 60 to 64: Reverse complement of the antisense oligonucleotides 42to 46 respectively; SEQ ID NO: 65 and 66: Assay sequences. LNA(2′-O,4′-C methylene locked nucleic acid): +A* or +T* or +C* or +G*;2′OM (2′-O-methyl): mU* or mA* or mC* or mG*; PS (phosphothioate): T* orA* or G* or c*; RNA: rU or rA or rG or rC.

DETAILED DESCRIPTION

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the ordering ofacts or events, as some acts may occur in different orders and/orconcurrently with other acts or events. Furthermore, not all illustratedacts or events are required to implement a methodology in accordancewith the present invention.

All genes, gene names, and gene products disclosed herein are intendedto correspond to homologs from any species for which the compositionsand methods disclosed herein are applicable. Thus, the terms include,but are not limited to genes and gene products from humans and mice. Itis understood that when a gene or gene product from a particular speciesis disclosed, this disclosure is intended to be exemplary only, and isnot to be interpreted as a limitation unless the context in which itappears clearly indicates. Thus, for example, for the genes disclosedherein, which in some embodiments relate to mammalian nucleic acid andamino acid sequences are intended to encompass homologous and/ororthologous genes and gene products from other animals including, butnot limited to other mammals, fish, amphibians, reptiles, and birds. Inan embodiment, the genes or nucleic acid sequences are human.

DEFINITIONS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

As used herein, the term “mRNA” means the presently known mRNAtranscript(s) of a targeted gene, and any further transcripts which maybe elucidated.

By “antisense oligonucleotides” or “antisense compound” is meant an RNAor DNA molecule that binds to another RNA or DNA (target RNA, DNA). Forexample, if it is an RNA oligonucleotide it binds to another RNA targetby means of RNA-RNA interactions and alters the activity of the targetRNA. An antisense oligonucleotide can upregulate or downregulateexpression and/or function of a particular polynucleotide. Thedefinition is meant to include any foreign RNA or DNA molecule which isuseful from a therapeutic, diagnostic, or other viewpoint. Suchmolecules include, for example, antisense RNA or DNA molecules,interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA,enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA,antisense oligomeric compounds, antisense oligonucleotides, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds that hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, partiallysingle-stranded, or circular oligomeric compounds.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. The term “oligonucleotide”, alsoincludes linear or circular oligomers of natural and/or modifiedmonomers or linkages, including deoxyribonucleosides, ribonucleosides,substituted and alpha-anomeric forms thereof, peptide nucleic acids(PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate,and the like. Oligonucleotides are capable of specifically binding to atarget polynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, Hoögsteen orreverse Hoögsteen types of base pairing, or the like.

The oligonucleotide may be “chimeric”, that is, composed of differentregions. In the context of this invention “chimeric” compounds areoligonucleotides, which contain two or more chemical regions, forexample, DNA region(s), RNA region(s), PNA region(s) etc. Each chemicalregion is made up of at least one monomer unit, i.e., a nucleotide inthe case of an oligonucleotides compound. These oligonucleotidestypically comprise at least one region wherein the oligonucleotide ismodified in order to exhibit one or more desired properties. The desiredproperties of the oligonucleotide include, but are not limited, forexample, to increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. Different regions of the oligonucleotide may thereforehave different properties. The chimeric oligonucleotides of the presentinvention can be formed as mixed structures of two or moreoligonucleotides, modified oligonucleotides, oligonucleosides and/oroligonucleotide analogs as described above.

The oligonucleotide can be composed of regions that can be linked in“register”, that is, when the monomers are linked consecutively, as innative DNA, or linked via spacers. The spacers are intended toconstitute a covalent “bridge” between the regions and have in preferredcases a length not exceeding about 100 carbon atoms. The spacers maycarry different functionalities, for example, having positive ornegative charge, carry special nucleic acid binding properties(intercalators, groove binders, toxins, fluorophors etc.), beinglipophilic, inducing special secondary structures like, for example,alanine containing peptides that induce alpha-helices.

As used herein “BDNF” and “Brain derived neurotrophic factor” areinclusive of all family members, mutants, alleles, fragments, species,coding and noncoding sequences, sense and antisense polynucleotidestrands, etc.

As used herein, the words ‘Brain derived neurotrophic factor’,‘Brain-derived neurotrophic factor’ and BDNF, are considered the same inthe literature and are used interchangeably in the present application.

As used herein, the term “oligonucleotide specific for” or“oligonucleotide which targets” refers to an oligonucleotide having asequence (i) capable of forming a stable complex with a portion of thetargeted gene, or (ii) capable of forming a stable duplex with a portionof a mRNA transcript of the targeted gene. Stability of the complexesand duplexes can be determined by theoretical calculations and/or invitro assays. Exemplary assays for determining stability ofhybridization complexes and duplexes are described in the Examplesbelow.

As used herein, the term “target nucleic acid” encompasses DNA, RNA(comprising premRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA, coding, noncoding sequences, sense or antisensepolynucleotides. The specific hybridization of an oligomeric compoundwith its target nucleic acid interferes with the normal function of thenucleic acid. This modulation of function of a target nucleic acid bycompounds, which specifically hybridize to it, is generally referred toas “antisense”. The functions of DNA to be interfered include, forexample, replication and transcription. The functions of RNA to beinterfered, include all vital functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in or facilitatedby the RNA. The overall effect of such interference with target nucleicacid function is modulation of the expression of an encoded product oroligonucleotides.

RNA interference “RNAi” is mediated by double stranded RNA (dsRNA)molecules that have sequence-specific homology to their “target” nucleicacid sequences. In certain embodiments of the present invention, themediators are 5-25 nucleotide “small interfering” RNA duplexes (siRNAs).The siRNAs are derived from the processing of dsRNA by an RNase enzymeknown as Dicer. siRNA duplex products are recruited into a multi-proteinsiRNA complex termed RISC (RNA Induced Silencing Complex). Withoutwishing to be bound by any particular theory, a RISC is then believed tobe guided to a target nucleic acid (suitably mRNA), where the siRNAduplex interacts in a sequence-specific way to mediate cleavage in acatalytic fashion. Small interfering RNAs that can be used in accordancewith the present invention can be synthesized and used according toprocedures that are well known in the art and that will be familiar tothe ordinarily skilled artisan. Small interfering RNAs for use in themethods of the present invention suitably comprise between about 1 toabout 50 nucleotides (nt). In examples of non limiting embodiments,siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt,about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25nucleotides.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs that automatically align nucleic acid sequences andindicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species. In the case of genes that have not been sequenced,Southern blots are performed to allow a determination of the degree ofidentity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of oligonucleotides thatexhibit a high degree of complementarity to target nucleic acidsequences in a subject to be controlled and a lower degree ofcomplementarity to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

By “enzymatic RNA” is meant an RNA molecule with enzymatic activity(Cech, (1988) J. American. Med. Assoc. 260, 3030-3035). Enzymaticnucleic acids (ribozymes) act by first binding to a target RNA. Suchbinding occurs through the target binding portion of an enzymaticnucleic acid which is held in close proximity to an enzymatic portion ofthe molecule that acts to cleave the target RNA. Thus, the enzymaticnucleic acid first recognizes and then binds a target RNA through basepairing, and once bound to the correct site, acts enzymatically to cutthe target RNA.

By “decoy RNA” is meant an RNA molecule that mimics the natural bindingdomain for a ligand. The decoy RNA therefore competes with naturalbinding target for the binding of a specific ligand. For example, it hasbeen shown that over-expression of HIV trans-activation response (TAR)RNA can act as a “decoy” and efficiently binds HIV tat protein, therebypreventing it from binding to TAR sequences encoded in the HIV RNA. Thisis meant to be a specific example. Those in the art will recognize thatthis is but one example, and other embodiments can be readily generatedusing techniques generally known in the art.

As used herein, the term “monomers” typically indicates monomers linkedby phosphodiester bonds or analogs thereof to form oligonucleotidesranging in size from a few monomeric units, e.g., from about 3-4, toabout several hundreds of monomeric units. Analogs of phosphodiesterlinkages include: phosphorothioate, phosphorodithioate,methylphosphonates, phosphoroselenoate, phosphoramidate, and the like,as more fully described below.

The term “nucleotide” covers naturally occurring nucleotides as well asnonnaturally occurring nucleotides. It should be clear to the personskilled in the art that various nucleotides which previously have beenconsidered “non-naturally occurring” have subsequently been found innature. Thus, “nucleotides” includes not only the known purine andpyrimidine heterocycles-containing molecules, but also heterocyclicanalogues and tautomers thereof. Illustrative examples of other types ofnucleotides are molecules containing adenine, guanine, thymine,cytosine, uracil, purine, xanthine, diaminopurine,8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine,N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine,isoguanin, inosine and the “non-naturally occurring” nucleotidesdescribed in Benner et al., U.S. Pat. No. 5,432,272. The term“nucleotide” is intended to cover every and all of these examples aswell as analogues and tautomers thereof. Especially interestingnucleotides are those containing adenine, guanine, thymine, cytosine,and uracil, which are considered as the naturally occurring nucleotidesin relation to therapeutic and diagnostic application in humans.Nucleotides include the natural 2′-deoxy and 2′-hydroxyl sugars, e.g.,as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman,San Francisco, 1992) as well as their analogs.

“Analogs” in reference to nucleotides includes synthetic nucleotideshaving modified base moieties and/or modified sugar moieties (see e.g.,described generally by Scheit, Nucleotide Analogs, John Wiley, New York,1980; Freier & Altmann, (1997) Nucl. Acid. Res., 25(22), 4429-4443,Toulmé, J. J., (2001) Nature Biotechnology 19:17-18; Manoharan M.,(1999) Biochemica et Biophysica Acta 1489:117-139; Freier S. M., (1997)Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery &Development, 3: 203-213, Herdewin P., (2000) Antisense & Nucleic AcidDrug Dev., 10:297-310); 2′-O, 3′-C-linked[3.2.0]bicycloarabinonucleosides. Such analogs include syntheticnucleotides designed to enhance binding properties, e.g., duplex ortriplex stability, specificity, or the like.

As used herein, “hybridization” means the pairing of substantiallycomplementary strands of oligomeric compounds. One mechanism of pairinginvolves hydrogen bonding, which may be Watson-Crick, Hoögsteen orreversed Hoögsteen hydrogen bonding, between complementary nucleoside ornucleotide bases (nucleotides) of the strands of oligomeric compounds.For example, adenine and thymine are complementary nucleotides whichpair through the formation of hydrogen bonds. Hybridization can occurunder varying circumstances.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays.

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated. In general, stringent hybridization conditionscomprise low concentrations (<0.15M) of salts with inorganic cationssuch as Na+ or K+ (i.e., low ionic strength), temperature higher than20° C.-25° C. below the Tm of the oligomeric compound:target sequencecomplex, and the presence of denaturants such as formamide,dimethylformamide, dimethyl sulfoxide, or the detergent sodium dodecylsulfate (SDS). For example, the hybridization rate decreases 1.1% foreach 1% formamide. An example of a high stringency hybridizationcondition is 0.1× sodium chloride-sodium citrate buffer (SSC)/0.1% (w/v)SDS at 60° C. for 30 minutes.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleotides on one or two oligomeric strands. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, said target nucleic acid being a DNA, RNA, oroligonucleotide molecule, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position. The oligomeric compound and the further DNA,RNA, or oligonucleotide molecule are complementary to each other when asufficient number of complementary positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of precise pairing or complementarityover a sufficient number of nucleotides such that stable and specificbinding occurs between the oligomeric compound and a target nucleicacid.

It is understood in the art that the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure). The oligomeric compounds of the presentinvention comprise at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 99% sequence complementarity to atarget region within the target nucleic acid sequence to which they aretargeted. For example, an antisense compound in which 18 of 20nucleotides of the antisense compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. In this example, the remainingnon-complementary nucleotides may be clustered or interspersed withcomplementary nucleotides and need not be contiguous to each other or tocomplementary nucleotides. As such, an antisense compound which is 18nucleotides in length having 4 (four) non-complementary nucleotideswhich are flanked by two regions of complete complementarity with thetarget nucleic acid would have 77.8% overall complementarity with thetarget nucleic acid and would thus fall within the scope of the presentinvention. Percent complementarity of an antisense compound with aregion of a target nucleic acid can be determined routinely using BLASTprograms (basic local alignment search tools) and PowerBLAST programsknown in the art. Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., (1981) 2, 482-489).

As used herein, the term “Thermal Melting Point (Tm)” refers to thetemperature, under defined ionic strength, pH, and nucleic acidconcentration, at which 50% of the oligonucleotides complementary to thetarget sequence hybridize to the target sequence at equilibrium.Typically, stringent conditions will be those in which the saltconcentration is at least about 0.01 to 1.0 M Na ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short oligonucleotides (e.g., 10 to 50 nucleotide). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

As used herein, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene.

The term “variant”, when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to a wild typegene. This definition may also include, for example, “allelic,”“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. Of particular utility in the invention are variants of wildtype gene products. Variants may result from at least one mutation inthe nucleic acid sequence and may result in altered mRNAs or inpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes that give rise to variants aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

The resulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs) or single base mutations in which thepolynucleotide sequence varies by one base. The presence of SNPs may beindicative of, for example, a certain population with a propensity for adisease state, that is susceptibility versus resistance.

Derivative polynucleotides include nucleic acids subjected to chemicalmodification, for example, replacement of hydrogen by an alkyl, acyl, oramino group. Derivatives, e.g., derivative oligonucleotides, maycomprise non-naturally-occurring portions, such as altered sugarmoieties or inter-sugar linkages. Exemplary among these arephosphorothioate and other sulfur containing species which are known inthe art. Derivative nucleic acids may also contain labels, includingradionucleotides, enzymes, fluorescent agents, chemiluminescent agents,chromogenic agents, substrates, cofactors, inhibitors, magneticparticles, and the like.

A “derivative” polypeptide or peptide is one that is modified, forexample, by glycosylation, pegylation, phosphorylation, sulfation,reduction/alkylation, acylation, chemical coupling, or mild formalintreatment. A derivative may also be modified to contain a detectablelabel, either directly or indirectly, including, but not limited to, aradioisotope, fluorescent, and enzyme label.

As used herein, the term “animal” or “patient” is meant to include, forexample, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys,horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken,reptiles, fish, insects and arachnids.

“Mammal” covers warm blooded mammals that are typically under medicalcare (e.g., humans and domesticated animals). Examples include feline,canine, equine, bovine, and human, as well as just human.

“Treating” or “treatment” covers the treatment of a disease-state in amammal, and includes: (a) preventing the disease-state from occurring ina mammal, in particular, when such mammal is predisposed to thedisease-state but has not yet been diagnosed as having it; (b)inhibiting the disease-state, e.g., arresting it development; and/or (c)relieving the disease-state, e.g., causing regression of the diseasestate until a desired endpoint is reached. Treating also includes theamelioration of a symptom of a disease (e.g., lessen the pain ordiscomfort), wherein such amelioration may or may not be directlyaffecting the disease (e.g., cause, transmission, expression, etc.).

As used herein, “cancer” refers to all types of cancer or neoplasm ormalignant tumors found in mammals, including, but not limited to:leukemias, lymphomas, melanomas, carcinomas and sarcomas. The cancermanifests itself as a “tumor” or tissue comprising malignant cells ofthe cancer. Examples of tumors include sarcomas and carcinomas such as,but not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. Additional cancers which can be treated by the disclosedcomposition according to the invention include but not limited to, forexample, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma,neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid, urinarybladder cancer, gastric cancer, premalignant skin lesions, testicularcancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,genitourinary tract cancer, malignant hypercalcemia, cervical cancer,endometrial cancer, adrenal cortical cancer, and prostate cancer.

As used herein a “Neurological disease or disorder” refers to anydisease or disorder of the nervous system and/or visual system.“Neurological disease or disorder” include disease or disorders thatinvolve the central nervous system (brain, brainstem and cerebellum),the peripheral nervous system (including cranial nerves), and theautonomic nervous system (parts of which are located in both central andperipheral nervous system). A Neurological disease or disorder includesbut is not limited to acquired epileptiform aphasia; acute disseminatedencephalomyelitis; adrenoleukodystrophy; age-related maculardegeneration; agenesis of the corpus callosum; agnosia; Aicardisyndrome; Alexander disease; Alpers' disease; alternating hemiplegia;Alzheimer's disease; Vascular dementia; amyotrophic lateral sclerosis;anencephaly; Angelman syndrome; angiomatosis; anoxia; aphasia; apraxia;arachnoid cysts; arachnoiditis; Anronl-Chiari malformation;arteriovenous malformation; Asperger syndrome; ataxia telegiectasia;attention deficit hyperactivity disorder; autism; autonomic dysfunction;back pain; Batten disease; Behcet's disease; Bell's palsy; benignessential blepharospasm; benign focal; amyotrophy; benign intracranialhypertension; Binswanger's disease; blepharospasm; Bloch Sulzbergersyndrome; brachial plexus injury; brain abscess; brain injury; braintumors (including glioblastoma multiforme); spinal tumor; Brown-Sequardsyndrome; Canavan disease; carpal tunnel syndrome; causalgia; centralpain syndrome; central pontine myelinolysis; cephalic disorder; cerebralaneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebralgigantism; cerebral palsy; Charcot-Marie-Tooth disease;chemotherapy-induced neuropathy and neuropathic pain; Chiarimalformation; chorea; chronic inflammatory demyelinating polyneuropathy;chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome;coma, including persistent vegetative state; congenital facial diplegia;corticobasal degeneration; cranial arteritis; craniosynostosis;Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing'ssyndrome; cytomegalic inclusion body disease; cytomegalovirus infection;dancing eyes-dancing feet syndrome; DandyWalker syndrome; Dawsondisease; De Morsier's syndrome; Dejerine-Klumke palsy; dementia;dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia;dysgraphia; dyslexia; dystonias; early infantile epilepticencephalopathy; empty sella syndrome; encephalitis; encephaloceles;encephalotrigeminal angiomatosis; epilepsy; Erb's palsy; essentialtremor; Fabry's disease; Fahr's syndrome; fainting; familial spasticparalysis; febrile seizures; Fisher syndrome; Friedreich's ataxia;fronto-temporal dementia and other “tauopathies”; Gaucher's disease;Gerstmann's syndrome; giant cell arteritis; giant cell inclusiondisease; globoid cell leukodystrophy; Guillain-Barre syndrome;HTLV-1-associated myelopathy; Hallervorden-Spatz disease; head injury;headache; hemifacial spasm; hereditary spastic paraplegia; heredopathiaatactic a polyneuritiformis; herpes zoster oticus; herpes zoster;Hirayama syndrome; HIV associated dementia and neuropathy (alsoneurological manifestations of AIDS); holoprosencephaly; Huntington'sdisease and other polyglutamine repeat diseases; hydranencephaly;hydrocephalus; hypercortisolism; hypoxia; immune-mediatedencephalomyelitis; inclusion body myositis; incontinentia pigmenti;infantile phytanic acid storage disease; infantile refsum disease;infantile spasms; inflammatory myopathy; intracranial cyst; intracranialhypertension; Joubert syndrome; Keams-Sayre syndrome; Kennedy diseaseKinsboume syndrome; Klippel Feil syndrome; Krabbe disease;Kugelberg-Welander disease; kuru; Lafora disease; Lambert-Eatonmyasthenic syndrome; Landau-Kleffner syndrome; lateral medullary(Wallenberg) syndrome; learning disabilities; Leigh's disease;Lennox-Gustaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy bodydementia; Lissencephaly; locked-in syndrome; Lou Gehrig's disease (i.e.,motor neuron disease or amyotrophic lateral sclerosis); lumbar discdisease; Lyme disease—neurological sequelae; Machado-Joseph disease;macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome; Menieresdisease; meningitis; Menkes disease; metachromatic leukodystrophy;microcephaly; migraine; Miller Fisher syndrome; mini-strokes;mitochondrial myopathies; Mobius syndrome; monomelic amyotrophy; motorneuron disease; Moyamoya disease; mucopolysaccharidoses; milti-infarctdementia; multifocal motor neuropathy; multiple sclerosis and otherdemyelinating disorders; multiple system atrophy with posturalhypotension; muscular dystrophy; myasthenia gravis; myelinoclasticdiffuse sclerosis; myoclonic encephalopathy of infants; myoclonus;myopathy; myotonia congenital; narcolepsy; neurofibromatosis;neuroleptic malignant syndrome; neurological manifestations of AIDS;neurological sequelae of lupus; neuromyotonia; neuronal ceroidlipofuscinosis; neuronal migration disorders; Niemann-Pick disease;O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinaldysraphism sequence; Ohtahara syndrome; olivopontocerebellar atrophy;opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overusesyndrome; paresthesia; a neurodegenerative disease or disorder(Parkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), dementia, multiple sclerosis andother diseases and disorders associated with neuronal cell death);paramyotonia congenital; paraneoplastic diseases; paroxysmal attacks;Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodicparalyses; peripheral neuropathy; painful neuropathy and neuropathicpain; persistent vegetative state; pervasive developmental disorders;photic sneeze reflex; phytanic acid storage disease; Pick's disease;pinched nerve; pituitary tumors; polymyositis; porencephaly; post-poliosyndrome; postherpetic neuralgia; postinfectious encephalomyelitis;postural hypotension; Prader-Willi syndrome; primary lateral sclerosis;prion diseases; progressive hemifacial atrophy; progressivemultifocalleukoencephalopathy; progressive sclerosing poliodystrophy;progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Huntsyndrome (types I and II); Rasmussen's encephalitis; reflex sympatheticdystrophy syndrome; Refsum disease; repetitive motion disorders;repetitive stress injuries; restless legs syndrome;retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome; SaintVitus dance; Sandhoff disease; Schilder's disease; schizencephaly;septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Dragersyndrome; Sjogren's syndrome; sleep apnea; Soto's syndrome; spasticity;spina bifida; spinal cord injury; spinal cord tumors; spinal muscularatrophy; Stiff-Person syndrome; stroke; Sturge-Weber syndrome; subacutesclerosing panencephalitis; subcortical arteriosclcrotic encephalopathy;Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachsdisease; temporal arteritis; tethered spinal cord syndrome; Thomsendisease; thoracic outlet syndrome; Tic Douloureux; Todd's paralysis;Tourette syndrome; transient ischemic attack; transmissible spongiformencephalopathies; transverse myelitis; traumatic brain injury; tremor;trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis;vascular dementia (multi-infarct dementia); vasculitis includingtemporal arteritis; Von Hippel-Lindau disease; Wallenberg's syndrome;Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome;Wildon's disease; and Zellweger syndrome.

A “proliferative disease or disorder” includes, but is not limited to,hematopoietic neoplastic disorders involving hyperplastic/neoplasticcells of hematopoietic origin arising from myeloid, lymphoid orerythroid lineages, or precursor cells thereof. These include, but arenot limited to erythroblastic leukemia, acute promyeloid leukemia(APML), chronic myelogenous leukemia (CML), lymphoid malignancies,including, but not limited to, acute lymphoblastic leukemia (ALL), whichincludes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia(CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to, non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

An “Inflammation” refers to systemic inflammatory conditions andconditions associated locally with migration and attraction ofmonocytes, leukocytes and/or neutrophils. Examples of inflammationinclude, but are not limited to, Inflammation resulting from infectionwith pathogenic organisms (including gram-positive bacteria,gram-negative bacteria, viruses, fungi, and parasites such as protozoaand helminths), transplant rejection (including rejection of solidorgans such as kidney, liver, heart, lung or cornea, as well asrejection of bone marrow transplants including graft-versus-host disease(GVHD)), or from localized chronic or acute autoimmune or allergicreactions. Autoimmune diseases include acute glomerulonephritis;rheumatoid or reactive arthritis; chronic glomerulonephritis;inflammatory bowel diseases such as Crohn's disease, ulcerative colitisand necrotizing enterocolitis; hepatitis; sepsis; alcoholic liverdisease; non-alcoholic steatosis; granulocyte transfusion associatedsyndromes; inflammatory dermatoses such as contact dermatitis, atopicdermatitis, psoriasis; systemic lupus erythematosus (SLE), autoimmunethyroiditis, multiple sclerosis, and some forms of diabetes, or anyother autoimmune state where attack by the subject's own immune systemresults in pathologic tissue destruction. Allergic reactions includeallergic asthma, chronic bronchitis, acute and delayed hypersensitivity.Systemic inflammatory disease states include inflammation associatedwith trauma, burns, reperfusion following ischemic events (e.g.thrombotic events in heart, brain, intestines or peripheral vasculature,including myocardial infarction and stroke), sepsis, ARDS or multipleorgan dysfunction syndrome. Inflammatory cell recruitment also occurs inatherosclerotic plaques. Inflammation includes, but is not limited to,Non-Hodgkin's lymphoma, Wegener's granulomatosis, Hashimoto'sthyroiditis, hepatocellular carcinoma, thymus atrophy, chronicpancreatitis, rheumatoid arthritis, reactive lymphoid hyperplasia,osteoarthritis, ulcerative colitis, papillary carcinoma, Crohn'sdisease, ulcerative colitis, acute cholecystitis, chronic cholecystitis,cirrhosis, chronic sialadenitis, peritonitis, acute pancreatitis,chronic pancreatitis, chronic Gastritis, adenomyosis, endometriosis,acute cervicitis, chronic cervicitis, lymphoid hyperplasia, multiplesclerosis, hypertrophy secondary to idiopathic thrombocytopenic purpura,primary IgA nephropathy, systemic lupus erythematosus, psoriasis,pulmonary emphysema, chronic pyelonephritis, and chronic cystitis.

Polynucleotide and Oligonucleotide Compositions and Molecules

Targets:

In one embodiment, the targets comprise nucleic acid sequences of Brainderived neurotrophic factor (BDNF), including without limitation senseand/or antisense noncoding and/or coding sequences associated with BDNF.PCT Pub. No. WO 2010/093904 and U.S. Pat. App. Pub. No. 2011/0319475,both titled “Treatment of Brain Derived Neurotrophic Factor (BDNF)Related Diseases by Inhibition of Natural Antisense Transcript to BDNF”and incorporated by reference herein in their entirety, disclose BDNF asa target for modulation using oligonucleotides as recited therein.

Neurotrophins are a class of structurally related growth factors thatpromote neural survival and differentiation. They stimulate neuriteoutgrowth, suggesting that they can promote regeneration of injuredneurons, and act as target-derived neurotrophic factors to stimulatecollateral sprouting in target tissues that produce the neurotrophi.Brain-derived neurotrophic factor (BDNF) was initially characterized asa basic protein present in brain extracts and capable of increasing thesurvival of dorsal root ganglia. When axonal communication with the cellbody is interrupted by injury, Schwann cells produce neurotrophicfactors such as nerve growth factor (NGF) and BDNF. Neurotrophins arereleased from the Schwann cells and dispersed diffusely in gradientfashion around regenerating axons, which then extend distally along theneurotrophins' density gradient. Local application of BDNF to transectednerves in neonatal rats has been shown to prevent massive death of motorneurons that follows axotomy. The mRNA titer of BDNF increases toseveral times the normal level four days after auxotomy and reaches itsmaximum at 4 weeks. Moreover, BDNF has been reported to enhance thesurvival of cholinergic neurons in culture.

In an embodiment, antisense oligonucleotides are used to prevent ortreat diseases or disorders associated with BDNF family members.Exemplary Brain derived neurotrophic factor (BDNF) mediated diseases anddisorders which can be treated with the antisense oligonucleotides ofthe invention and/or with cell/tissues regenerated from stem cellsobtained using and/or having the antisense compounds comprise: a diseaseor disorder associated with abnormal function and/or expression of BDNF,a neurological disease or disorder, a disease or a disorder associatedwith defective neurogenesis; a neurodegenerative disease or disorder(e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis etc.); a neuropsychiatric disorder(depression, schizophrenia, schizofreniform disorder, schizoaffectivedisorder, and delusional disorder; anxiety disorders such as panicdisorder, phobias (including agoraphobia), an obsessive-compulsivedisorder, a posttraumatic stress disorder, a bipolar disorder, anorexianervosa, bulimia nervosa), an autoimmune disorder (e.g., multiplesclerosis) of the central nervous system, memory loss, a long term or ashort term memory disorder, benign forgetfulness, a childhood learningdisorder, close head injury, an attention deficit disorder, neuronalreaction to viral infection, brain damage, narcolepsy, a sleep disorder(e.g., circadian rhythm disorders, insomnia and narcolepsy); severanceof nerves or nerve damage, severance of cerebrospinal nerve cord (CNS)and a damage to brain or nerve cells, a neurological deficit associatedwith AIDS, a motor and tic disorder characterized by motor and/or vocaltics (e.g., Tourette's disorder, chronic motor or vocal tic disorder,transient tic disorder, and stereotypic movement disorder), a substanceabuse disorder (e.g., substance dependence, substance abuse and thesequalae of substance abuse/dependence, such as substance-inducedpsychological disorder, substance withdrawal and substance-induceddementia or amnestic disorder), traumatic brain injury, tinnitus,neuralgia (e.g., trigeminal neuralgia) pain (e.g chronic pain, chronicinflammatory pain, pain associated with arthritis, fibromyalgia, backpain, cancer-associated pain, pain associated with digestive disease,pain associated with Crohn's disease, pain associated with autoimmunedisease, pain associated with endocrine disease, pain associated withdiabetic neuropathy, phantom limb pain, spontaneous pain, chronicpost-surgical pain, chronic temporomandibular pain, causalgia,post-herpetic neuralgia, AIDS-related pain, complex regional painsyndromes type I and II, trigeminal neuralgia, chronic back pain, painassociated with spinal cord injury, pain associated with drug intake andrecurrent acute pain, neuropathic pain), inappropriate neuronal activityresulting in neurodysthesias in a disease such as diabetes, an MS and amotor neuron disease, ataxias, muscular rigidity (spasticity),temporomandibular joint dysfunction, Reward deficiency syndrome (RDS),neurotoxicity caused by alcohol or substance abuse (e.g., ecstasy,methamphetamine etc.), mental retardation or cognitive impairment (e.g.,nonsyndromic X-linked mental retardation, fragile X syndrome, Down'ssyndrome, autism), aphasia, Bell's palsy, Creutzfeldt-jacob disease,encephalitis, age related macular degeneration, ondine syndrome, WAGRsyndrome, hearing loss, Rett syndrome, epilepsy, spinal cord injury,stroke, hypoxia, ischemia, brain injury, diabetic neuropathy, peripheralneuropathy, nerve transplantation complications, motor neuron disease,peripheral nerve injury, obesity, a metabolic syndrome, cancer, asthma,an atopic disease, inflammation, allergy, eczema, a neuro-oncologicaldisease or disorder, neuro-immunological disease or disorder andneuro-otological disease or disorder; and a disease or disorderassociated with aging and senescence.

The present invention provides a mechanism by which endogenous NATssuppress transcription of their sense gene counterparts. The inventionprovides that endogenous gene expression can be upregulated, in a locusspecific manner by the removal or inhibition of the NATs, which aretranscribed from most transcriptional units.

One embodiment of the present invention provides examples of functionalncRNAs that regulate protein output, the phenomenon applicable to manyother genomic loci.

The Brain-derived Neurotrophic Factor (BDNF) is a member of the“neurotrophin” family of growth factors, essential for neuronal growth,maturation differentiation and maintenance. BDNF is also essential forneuronal plasticity and shown to be involved in learning, and memoryprocesses. The BDNF locus is on chromosome 11 and shows activetranscription from both strands, which leads to transcription of anoncoding NATs.

The present invention characterizes the regulatory role of thisantisense RNA molecule, BDNF-AS that exerts a potent reciprocal anddynamic regulation over the expression of sense BDNF mRNA and protein,both in vitro and in vivo.

One embodiment of the present invention provides a strategy forupregulation of mRNA expression, using antisense RNA transcriptinhibitory molecules, which are termed as AntagoNATs. AntagoNATs aredescribed, e.g., in PCT Pub. No. WO 2012/068340, incorporated herein byreference in its entirety.

The number of ncRNAs in eukaryotic genomes have been shown to increaseas a function of developmental complexity and there is, for example, agreat deal of diversity in ncRNAs expressed in the nervous system. Overthe past few years, there have been reports on functional NATs andshowed their potential involvement in human disorders, includingAlzheimer's disease, Parkinson's disease and Fragile X syndrome.Moreover, it has been reported that upregulation of CD97 sense gene canbe attained by knockdown of its antisense RNA transcript. Upregulationof progesterone receptor (PR), and other endogenous transcripts wasreported following targeting of promoter-derived noncoding RNAs.Transcriptional activation of p21 gene and Oct4 promoter were reportedfollowing NATs depletion. Antisense RNA-induced chromatin remodelingseems to be a feasible and dynamic mode of action for many low copynumber NATs. If so, antisense RNA might predominantly exert localeffects to maintain or modify chromatin structure, ultimately activatingor suppressing sense gene expression.

PCR2 is a protein complex that consists of four core subunits: Ecd,Suz12, RbAp48 and the catalytic Ezh2, that catalyzes the trimethylationof histone H3-lysine. (H3K27met3). Recent studies provide evidence fordirect RNA-potein interaction between Ezh2 and many ncRNA transcripts.Other studies of X inactivation and HOX gene cluster show RNAtranscripts to be involved in the PRC2-mediated induction of H3K27met3,repressive chromatin marks. PRC2 transcriptome profiling has identifiedover 9,000 PRC2-interacting RNAs in embryonic stem cells, many of themcategorized as antisense RNA transcripts. Epigenetic silencing of p15and DM1 genes were reported to involve heterochromatin formation by itsantisense RNA. The traditional binary division of chromatin into hetero-or eu-chromatin categories might not be complete as recent work hasshown that there are five principal chromatin types that are moredynamic and flexible than originally believed. Likely applicable to alarge number of gene loci, NATs can be manipulated in order to obtain alocus-specific alteration in chromatin modification. As examples, it isshown that cleavage (by siRNA) or inhibition (by AntagoNATs) of theantisense transcripts of BDNF genes leads to the upregulation ofcorresponding mRNAs.

Neurotrophins belong to a class of secreted growth factors that enhancethe survival, development, differentiation and function of neurons andBDNF is an important molecular mediator of synaptic plasticity. BDNF issuggested to synchronize neuronal and glial maturation, participate inaxonal and dendritic differentiation and protect and enhance neuronalcell survival. Neurotrophin expression levels are impaired inneurodegenerative and in psychiatric and neurodevelopmental disorders.The upregulation of neurotrophins is believed to have beneficial effectson several neurological disorders. AntagoNATs can be used as atherapeutic strategy to inhibit BDNF-AS and consequently enhanceneuronal proliferation and survival in a variety of disease states. Itcannot be excluded that the herein described approach to upregulate thesynthesis of endogenous BDNF molecules, presumed to contain naturalmodifications and to represent all known splice forms, will prove to bedistinct, and perhaps superior, to administrating synthetic BDNFmolecules.

In an embodiment, modulation of BDNF by one or more antisenseoligonucleotides is administered to a patient in need thereof, toprevent or treat any disease or disorder related to BDNF abnormalexpression, function, activity as compared to a normal control.

In an embodiment, the oligonucleotides are specific for naturalantisense transcripts of BDNF recited herein, which includes, withoutlimitation noncoding regions. The BDNF targets comprise variants ofBDNF; mutants of BDNF, including SNPs; noncoding sequences of BDNF;alleles, fragments and the like. Preferably the oligonucleotide is anantisense RNA molecule.

In accordance with embodiments of the invention, the target nucleic acidmolecule is not limited to BDNF polynucleotides alone but extends to anyof the isoforms, receptors, homologs, non-coding regions and the like ofBDNF.

In an embodiment, an oligonucleotide targets a natural antisensesequence (natural antisense to the coding and non-coding regions) ofBDNF targets, including, without limitation, variants, alleles,homologs, mutants, derivatives, fragments and complementary sequencesthereto. Preferably the oligonucleotide is an antisense RNA or DNAmolecule.

In an embodiment, the oligomeric compounds of the present invention alsoinclude variants in which a different base is present at one or more ofthe nucleotide positions in the compound. For example, if the firstnucleotide is an adenine, variants may be produced which containthymidine, guanosine, cytidine or other natural or unnatural nucleotidesat this position. This may be done at any of the positions of theantisense compound. These compounds are then tested using the methodsdescribed herein to determine their ability to inhibit expression of atarget nucleic acid.

In some embodiments, homology, sequence identity or complementarity,between the antisense compound and target is from about 50% to about60%. In some embodiments, homology, sequence identity orcomplementarity, is from about 60% to about 70%. In some embodiments,homology, sequence identity or complementarity, is from about 70% toabout 80%. In some embodiments, homology, sequence identity orcomplementarity, is from about 80% to about 90%. In some embodiments,homology, sequence identity or complementarity, is about 90%, about 92%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100%.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired. Such conditionsinclude, i.e., physiological conditions in the case of in vivo assays ortherapeutic treatment, and conditions in which assays are performed inthe case of in vitro assays.

An antisense compound, whether DNA, RNA, chimeric, substituted etc, isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarily to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed.

In an embodiment, targeting of BDNF including without limitation,antisense sequences which are identified and expanded, using forexample, PCR, hybridization etc., one or more of the sequences set forthas SEQ ID NOS: 3 to 11, and the like, modulate the expression orfunction of BDNF. In one embodiment, expression or function isup-regulated as compared to a control. In an embodiment, expression orfunction is down-regulated as compared to a control.

In an embodiment, oligonucleotides comprise nucleic acid sequences setforth as SEQ ID NOS: 12 to 49 including antisense sequences which areidentified and expanded, using for example, PCR, hybridization etc.These oligonucleotides can comprise one or more modified nucleotides,shorter or longer fragments, modified bonds and the like. Examples ofmodified bonds or internucleotide linkages comprise phosphorothioate,phosphorodithioate or the like. In an embodiment, the nucleotidescomprise a phosphorus derivative. The phosphorus derivative (or modifiedphosphate group) which may be attached to the sugar or sugar analogmoiety in the modified oligonucleotides of the present invention may bea monophosphate, diphosphate, triphosphate, alkylphosphate,alkanephosphate, phosphorothioate and the like. The preparation of theabove-noted phosphate analogs, and their incorporation into nucleotides,modified nucleotides and oligonucleotides, per se, is also known andneed not be described here.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotides have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

In embodiments of the present invention oligomeric antisense compounds,particularly oligonucleotides, bind to target nucleic acid molecules andmodulate the expression and/or function of molecules encoded by a targetgene. The functions of DNA to be interfered comprise, for example,replication and transcription. The functions of RNA to be interferedcomprise all vital functions such as, for example, translocation of theRNA to the site of protein translation, translation of protein from theRNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The functions may be up-regulated or inhibited depending on thefunctions desired.

The antisense compounds, include, antisense oligomeric compounds,antisense oligonucleotides, external guide sequence (EGS)oligonucleotides, alternate splicers, primers, probes, and otheroligomeric compounds that hybridize to at least a portion of the targetnucleic acid. As such, these compounds may be introduced in the form ofsingle-stranded, double-stranded, partially single-stranded, or circularoligomeric compounds.

Targeting an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes Brain derived neurotrophicfactor (BDNF).

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

In an embodiment, the antisense oligonucleotides bind to the naturalantisense sequences of Brain derived neurotrophic factor (BDNF) andmodulate the expression and/or function of BDNF (SEQ ID NO: 1 and 2).Examples of antisense sequences include SEQ ID NOS: 3 to 55.

In an embodiment, the antisense oligonucleotides bind to one or moresegments of Brain derived neurotrophic factor (BDNF) polynucleotides andmodulate the expression and/or function of BDNF. The segments compriseat least five consecutive nucleotides of the BDNF sense or antisensepolynucleotides.

In an embodiment, the antisense oligonucleotides are specific fornatural antisense sequences of BDNF wherein binding of theoligonucleotides to the natural antisense sequences of BDNF modulateexpression and/or function of BDNF.

In an embodiment, oligonucleotide compounds comprise sequences set forthas SEQ ID NOS: 12 to 49, antisense sequences which are identified andexpanded, using for example, PCR, hybridization etc Theseoligonucleotides can comprise one or more modified nucleotides, shorteror longer fragments, modified bonds and the like. Examples of modifiedbonds or internucleotide linkages comprise phosphorothioate,phosphorodithioate or the like. In an embodiment, the nucleotidescomprise a phosphorus derivative. The phosphorus derivative (or modifiedphosphate group) which may be attached to the sugar or sugar analogmoiety in the modified oligonucleotides of the present invention may bea monophosphate, diphosphate, triphosphate, alkylphosphate,alkanephosphate, phosphorothioate and the like. The preparation of theabove-noted phosphate analogs, and their incorporation into nucleotides,modified nucleotides and oligonucleotides, per se, is also known andneed not be described here.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes has a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG; and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formyl methionine (in prokaryotes).Eukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA transcribed from a geneencoding Brain derived neurotrophic factor (BDNF), regardless of thesequence(s) of such codons. A translation termination codon (or “stopcodon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAGand 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions that may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, atargeted region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Another target region includes the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene). Still another target regionincludes the 3′ untranslated region (3′UTR), known in the art to referto the portion of an mRNA in the 3′ direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3′ end of an mRNA (or correspondingnucleotides on the gene). The 5′ cap site of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap site. Another target region for thisinvention is the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. In one embodiment, targeting splicesites, i.e., intron-exon junctions or exon-intron junctions, isparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular splice product isimplicated in disease. An aberrant fusion junction due to rearrangementor deletion is another embodiment of a target site. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources are known as “fusion transcripts”. Introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

In an embodiment, the antisense oligonucleotides bind to coding and/ornon-coding regions of a target polynucleotide and modulate theexpression and/or function of the target molecule.

In an embodiment, the antisense oligonucleotides bind to naturalantisense polynucleotides and modulate the expression and/or function ofthe target molecule.

In an embodiment, the antisense oligonucleotides bind to sensepolynucleotides and modulate the expression and/or function of thetarget molecule.

Alternative RNA transcripts can be produced from the same genomic regionof DNA. These alternative transcripts are generally known as “variants”.More specifically, “pre-mRNA variants” are transcripts produced from thesame genomic DNA that differ from other transcripts produced from thesame genomic DNA in either their start or stop position and contain bothintronic and exonic sequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

Variants can be produced through the use of alternative signals to startor stop transcription. Pre-mRNAs and mRNAs can possess more than onestart codon or stop codon. Variants that originate from a pre-mRNA ormRNA that use alternative start codons are known as “alternative startvariants” of that pre-mRNA or mRNA. Those transcripts that use analternative stop codon are known as “alternative stop variants” of thatpre-mRNA or mRNA. One specific type of alternative stop variant is the“polyA variant” in which the multiple transcripts produced result fromthe alternative selection of one of the “polyA stop signals” by thetranscription machinery, thereby producing transcripts that terminate atunique polyA sites. Within the context of the invention, the types ofvariants described herein are also embodiments of target nucleic acids.

The locations on the target nucleic acid to which the antisensecompounds hybridize are defined as at least a 5-nucleotide long portionof a target region to which an active antisense compound is targeted.

While the specific sequences of certain exemplary target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional target segments are readilyidentifiable by one having ordinary skill in the art in view of thisdisclosure.

Target segments 5-100 nucleotides in length comprising a stretch of atleast five (5) consecutive nucleotides selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 5 consecutive nucleotides from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleotides beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 5 to about 100 nucleotides). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 5 consecutive nucleotides from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleotides being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 5 to about 100nucleotides). One having skill in the art armed with the target segmentsillustrated herein will be able, without undue experimentation, toidentify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

In embodiments of the invention the oligonucleotides bind to anantisense strand of a particular target. The oligonucleotides are atleast 5 nucleotides in length and can be synthesized so eacholigonucleotide targets overlapping sequences such that oligonucleotidesare synthesized to cover the entire length of the target polynucleotide.The targets also include coding as well as non coding regions.

In one embodiment, it is preferred to target specific nucleic acids byantisense oligonucleotides. Targeting an antisense compound to aparticular nucleic acid is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a non coding polynucleotidesuch as for example, non coding RNA (ncRNA).

RNAs can be classified into (1) messenger RNAs (mRNAs), which aretranslated into proteins, and (2) non-protein-coding RNAs (ncRNAs).ncRNAs comprise microRNAs, antisense transcripts and otherTranscriptional Units (TU) containing a high density of stop codons andlacking any extensive “Open Reading Frame”. Many ncRNAs appear to startfrom initiation sites in 3′ untranslated regions (3′UTRs) ofprotein-coding loci. ncRNAs are often rare and at least half of thencRNAs that have been sequenced by the FANTOM consortium seem not to bepolyadenylated. Most researchers have for obvious reasons focused onpolyadenylated mRNAs that are processed and exported to the cytoplasm.Recently, it was shown that the set of non-polyadenylated nuclear RNAsmay be very large, and that many such transcripts arise from so-calledintergenic regions. The mechanism by which ncRNAs may regulate geneexpression is by base pairing with target transcripts. The RNAs thatfunction by base pairing can be grouped into (1) cis encoded RNAs thatare encoded at the same genetic location, but on the opposite strand tothe RNAs they act upon and therefore display perfect complementarity totheir target, and (2) trans-encoded RNAs that are encoded at achromosomal location distinct from the RNAs they act upon and generallydo not exhibit perfect base-pairing potential with their targets.

Without wishing to be bound by theory, perturbation of an antisensepolynucleotide by the antisense oligonucleotides described herein canalter the expression of the corresponding sense messenger RNAs. However,this regulation can either be discordant (antisense knockdown results inmessenger RNA elevation) or concordant (antisense knockdown results inconcomitant messenger RNA reduction). In these cases, antisenseoligonucleotides can be targeted to overlapping or non-overlapping partsof the antisense transcript resulting in its knockdown or sequestration.Coding as well as non-coding antisense can be targeted in an identicalmanner and that either category is capable of regulating thecorresponding sense transcripts—either in a concordant or disconcordantmanner. The strategies that are employed in identifying newoligonucleotides for use against a target can be based on the knockdownof antisense RNA transcripts by antisense oligonucleotides or any othermeans of modulating the desired target.

Strategy 1:

In the case of discordant regulation, knocking down the antisensetranscript elevates the expression of the conventional (sense) gene.Should that latter gene encode for a known or putative drug target, thenknockdown of its antisense counterpart could conceivably mimic theaction of a receptor agonist or an enzyme stimulant.

Strategy 2:

In the case of concordant regulation, one could concomitantly knock downboth antisense and sense transcripts and thereby achieve synergisticreduction of the conventional (sense) gene expression. If, for example,an antisense oligonucleotide is used to achieve knockdown, then thisstrategy can be used to apply one antisense oligonucleotide targeted tothe sense transcript and another antisense oligonucleotide to thecorresponding antisense transcript, or a single energetically symmetricantisense oligonucleotide that simultaneously targets overlapping senseand antisense transcripts.

According to the present invention, antisense compounds includeantisense oligonucleotides, ribozymes, external guide sequence (EGS)oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, and otheroligomeric compounds which hybridize to at least a portion of the targetnucleic acid and modulate its function. As such, they may be DNA, RNA,DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one ormore of these. These compounds may be single-stranded, doublestranded,circular or hairpin oligomeric compounds and may contain structuralelements such as internal or terminal bulges, mismatches or loops.Antisense compounds are routinely prepared linearly but can be joined orotherwise prepared to be circular and/or branched. Antisense compoundscan include constructs such as, for example, two strands hybridized toform a wholly or partially double-stranded compound or a single strandwith sufficient self-complementarity to allow for hybridization andformation of a fully or partially double-stranded compound. The twostrands can be linked internally leaving free 3′ or 5′ termini or can belinked to form a continuous hairpin structure or loop. The hairpinstructure may contain an overhang on either the 5′ or 3′ terminusproducing an extension of single stranded character. The double strandedcompounds optionally can include overhangs on the ends. Furthermodifications can include conjugate groups attached to one of thetermini, selected nucleotide positions, sugar positions or to one of theinternucleoside linkages. Alternatively, the two strands can be linkedvia a non-nucleic acid moiety or linker group. When formed from only onestrand, dsRNA can take the form of a self-complementary hairpin-typemolecule that doubles back on itself to form a duplex. Thus, the dsRNAscan be fully or partially double stranded. Specific modulation of geneexpression can be achieved by stable expression of dsRNA hairpins intransgenic cell lines, however, in some embodiments, the gene expressionor function is up regulated. When formed from two strands, or a singlestrand that takes the form of a self-complementary hairpin-type moleculedoubled back on itself to form a duplex, the two strands (orduplex-forming regions of a single strand) are complementary RNA strandsthat base pair in Watson-Crick fashion.

Once introduced to a system, the compounds of the invention may elicitthe action of one or more enzymes or structural proteins to effectcleavage or other modification of the target nucleic acid or may workvia occupancy-based mechanisms. In general, nucleic acids (includingoligonucleotides) may be described as “DNA-like” (i.e., generally havingone or more 2′-deoxy sugars and, generally, T rather than U bases) or“RNA-like” (i.e., generally having one or more 2′-hydroxyl or2′-modified sugars and, generally U rather than T bases). Nucleic acidhelices can adopt more than one type of structure, most commonly the A-and B-forms. It is believed that, in general, oligonucleotides whichhave B-form-like structure are “DNA-like” and those which haveA-formlike structure are “RNA-like.” In some (chimeric) embodiments, anantisense compound may contain both A- and B-form regions.

In an embodiment, the desired oligonucleotides or antisense compounds,comprise at least one of: antisense RNA, antisense DNA, chimericantisense oligonucleotides, antisense oligonucleotides comprisingmodified linkages, interference RNA (RNAi), short interfering RNA(siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA(stRNA); or a short, hairpin RNA (shRNA); small RNA-induced geneactivation (RNAa); small activating RNAs (saRNAs), or combinationsthereof.

dsRNA can also activate gene expression, a mechanism that has beentermed “small RNA-induced gene activation” or RNAa. dsRNAs targetinggene promoters induce potent transcriptional activation of associatedgenes. RNAa was demonstrated in human cells using synthetic dsRNAs,termed “small activating RNAs” (saRNAs). It is currently not knownwhether RNAa is conserved in other organisms.

Small double-stranded RNA (dsRNA), such as small interfering RNA (siRNA)and microRNA (miRNA), have been found to be the trigger of anevolutionary conserved mechanism known as RNA interference (RNAi). RNAiinvariably leads to gene silencing via remodeling chromatin to therebysuppress transcription, degrading complementary mRNA, or blockingprotein translation. However, in instances described in detail in theexamples section which follows, oligonucleotides are shown to increasethe expression and/or function of the Brain derived neurotrophic factor(BDNF) polynucleotides and encoded products thereof. dsRNAs may also actas small activating RNAs (saRNA). Without wishing to be bound by theory,by targeting sequences in gene promoters, saRNAs would induce targetgene expression in a phenomenon referred to as dsRNA-inducedtranscriptional activation (RNAa).

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of Brain derived neurotrophic factor (BDNF)polynucleotides. “Modulators” are those compounds that decrease orincrease the expression of a nucleic acid molecule encoding BDNF andwhich comprise at least a 5-nucleotide portion that is complementary toa preferred target segment. The screening method comprises the steps ofcontacting a preferred target segment of a nucleic acid moleculeencoding sense or natural antisense polynucleotides of BDNF with one ormore candidate modulators, and selecting for one or more candidatemodulators which decrease or increase the expression of a nucleic acidmolecule encoding BDNF polynucleotides, e.g. SEQ ID NOS: 12 to 49. Onceit is shown that the candidate modulator or modulators are capable ofmodulating (e.g. either decreasing or increasing) the expression of anucleic acid molecule encoding BDNF polynucleotides, the modulator maythen be employed in further investigative studies of the function ofBDNF polynucleotides, or for use as a research, diagnostic, ortherapeutic agent in accordance with the present invention.

Targeting the natural antisense sequence preferably modulates thefunction of the target gene. For example, the BDNF gene (e.g. accessionnumber NM_(—)170735 and NM_(—)007540). In an embodiment, the target isan antisense polynucleotide of the BDNF gene. In an embodiment, anantisense oligonucleotide targets sense and/or natural antisensesequences of BDNF polynucleotides (e.g. accession number NM_(—)170735and NM_(—)007540), variants, alleles, isoforms, homologs, mutants,derivatives, fragments and complementary sequences thereto. Preferablythe oligonucleotide is an antisense molecule and the targets includecoding and noncoding regions of antisense and/or sense BDNFpolynucleotides.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocessing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications. For example, suchdouble-stranded moieties have been shown to inhibit the target by theclassical hybridization of antisense strand of the duplex to the target,thereby triggering enzymatic degradation of the target.

In an embodiment, an antisense oligonucleotide targets Brain derivedneurotrophic factor (BDNF) polynucleotides (e.g. accession numberNM_(—)170735 and NM_(—)007540), variants, alleles, homologs, mutants,derivatives, fragments and complementary sequences thereto. Preferablythe oligonucleotide is an antisense molecule.

In accordance with embodiments of the invention, the target nucleic acidmolecule is not limited to BDNF alone but extends to any polynucleotidevariant thereof and any polynucleotide that produces, affects, impactsor results in or relates to a BDNF expression product and/or anyisoforms thereof.

In an embodiment, an oligonucleotide targets a natural antisensesequence of BDNF polynucleotides, for example, polynucleotides set forthas SEQ ID NOS: 3 to 11, and any variants, alleles, homologs, mutants,derivatives, fragments and complementary sequences thereto. Examples ofantisense oligonucleotides are set forth as SEQ ID NOS: 12 to 49.

In one embodiment, the oligonucleotides are complementary to or bind tonucleic acid sequences of BDNF antisense, including without limitationnoncoding sense and/or antisense sequences associated with BDNFpolynucleotides and modulate expression and/or function of BDNFmolecules.

In an embodiment, the oligonucleotides are complementary to or bind tonucleic acid sequences of BDNF natural antisense, set forth as SEQ IDNOS: 3 to 11, and modulate expression and/or function of BDNF molecules.

In an embodiment, oligonucleotides comprise sequences of at least 5consecutive nucleotides of SEQ ID NOS: 12 to 49 and modulate expressionand/or function of BDNF molecules.

The polynucleotide targets comprise BDNF, including family membersthereof, variants of BDNF; mutants of BDNF, including SNPs; noncodingsequences of BDNF; alleles of BDNF; species variants, fragments and thelike. Preferably the oligonucleotide is an antisense molecule.

In an embodiment, the oligonucleotide targeting BDNF polynucleotides,comprise: antisense RNA, interference RNA (RNAi), short interfering RNA(siRNA); micro interfering RNA (miRNA); a small, temporal RNA (stRNA);or a short, hairpin RNA (shRNA); small RNA-induced gene activation(RNAa); or, small activating RNA (saRNA).

In an embodiment, targeting of Brain derived neurotrophic factor (BDNF)polynucleotides, e.g. SEQ ID NOS: 3 to 55 modulate the expression orfunction of these targets. In one embodiment, expression or function isup-regulated as compared to a control. In an embodiment, expression orfunction is down-regulated as compared to a control.

In an embodiment, antisense compounds comprise sequences set forth asSEQ ID NOS: 12 to 49. These oligonucleotides can comprise one or moremodified nucleotides, shorter or longer fragments, modified bonds andthe like.

In an embodiment, SEQ ID NOS: 12 to 49 comprise one or more LNAnucleotides. Table 1 shows exemplary antisense oligonucleotides usefulin the methods of the invention.

TABLE 1 Antisense Sequence ID Sequence Name Sequence SEQ ID NO: 12CUR-2046 ArArCrArArArCrArArCrUrGrGrUrGrArGrCrCrUrGrG (Antisense)SEQ ID NO: 13 CUR-2047 rUrGrArGrCrCrUrArArGrArUrArCrArUrUrGrCrUrCrUAntisense) SEQ ID NO: 14 CUR-2048rGrUrGrCrUrGrUrUrGrUrArArGrArUrUrArGrCrCrArC (Antisense) SEQ ID NO: 15CUR-2049 rArArUrGrArCrArUrGrUrUrUrGrUrArGrGrGrArGrCrC (Antisense)SEQ ID NO: 16 CUR-2050 +C*mC*mA*+G*mG*mU*+G*mU*mG*mC*+G*mG*mA*+CSEQ ID NO: 17 CUR-2051 +C*mC*mA*+U*mG*mG*+G*mA*mC*mU*+C*mU*mG*+GSEQ ID NO: 18 CUR-2052 +A*mG*mA*+G*mC*mG*+U*mG*mA*mA*+U*mG*mG+GSEQ ID NO: 19 CUR-2053 +C*mC*mC*+A*mA*mG*+G*mC*mA*mG*+G*mU*mU+CSEQ ID NO: 20 CUR-2054 +A*mA*mG*+A*mU*mG*+C*mU*mU*mG*+A*mC*mA*+USEQ ID NO: 21 CUR-2055 +C*mA*mU*+U*mG*mG*+C*mU*mG*mA*+C*mA*mC*+USEQ ID NO: 22 CUR-2056 +U*mU*mC*+G*mA*mA*+C*mA*mC*mG*+U*mG*mA*+USEQ ID NO: 23 CUR-2057 +A*mG*mA*+A*mG*mA*+G*mC*mU*mG*+U*mU*mG*+GSEQ ID NO: 24 CUR-2058 +A*mU*mG*+A*mG*mG*+A*mC*mC*mA*+G*mA*mA*+ASEQ ID NO: 25 CUR-2059 +G*mU*mU*+C*mG*mG*+C*mC*mC*mA*+A*mU*mG+ASEQ ID NO: 26 CUR-2060 +A*mG*mA*+A*mA*mA*+C*mA*mA*mU*+A*mA*mG*+GSEQ ID NO: 27 CUR-2061 +A*mC*mG*+C*mA*mG*+A*mC*mU*mU*+G*mU*mA+CSEQ ID NO: 28 CUR-2062 +A*mC*mG*+U*mC*mC*+A*mG*mG*mG*+U*mG*mA+USEQ ID NO: 29 CUR-2063 +G*mC*mU*+C*mA*mG*+U*mA*mG*mU*+C*mA*mA*+GSEQ ID NO: 30 CUR-2064 +U*mG*mC*+C*mU*mU*+U*mG*mG*mA*+G*mC*mC+USEQ ID NO: 31 CUR-2065 +C*mC*mU*+C*mU*mU*+C*mU*mC*mU*+U*mU*mC*+USEQ ID NO: 32 CUR-2066 +C*+C*+C*G*G*T*A*T*C*C*A*A*A*+G*+G*+CSEQ ID NO: 33 CUR-2067 +G*+T*+A*T*T*A*G*C*G*A*G*T*G*+G*+G*+TSEQ ID NO: 34 CUR-2068 +G*+T*+C*T*A*T*G*A*G*G*G*T*T*+C*+G*+GSEQ ID NO: 35 CUR-2069 +C*+C*+T*C*C*T*C*T*A*C*T*C*T*+T*+T*+CSEQ ID NO: 36 CUR-2070 +G*+G*+C*A*G*G*T*T*C*G*A*G*A*+G*+G*+TSEQ ID NO: 37 CUR-2071 +T*+T*+C*C*T*T*C*C*C*A*C*A*G*+T*+T*+CSEQ ID NO: 38 CUR-2072 +C*+G*+G*T*T*G*C*A*T*G*A*A*G*+G*+C*+GSEQ ID NO: 39 CUR-2073 +T*+G*+G*C*T*G*G*C*G*A*T*T*C*+A*+T*+ASEQ ID NO: 40 CUR-2074 +C*+A*+A*C*A*T*A*T*C*A*G*G*A*+G*+C*+CSEQ ID NO: 41 CUR-2075 +T*+G*+T*A*T*T*C*C*C*A*G*A*A*+C*+T*+TSEQ ID NO: 42 CUR-2076rUrArUrGrGrUrUrArUrUrUrCrArUrArCrUrUrCrGrGrUrUrGrCrArUrG (Antisense)SEQ ID NO: 43 CUR-2077rArGrArArGrUrArArArCrGrUrCrCrArCrGrGrArCrArArGrGrCrArArC (Antisense)SEQ ID NO: 44 CUR-2078rArUrUrUrCrUrArCrGrArGrArCrCrArArGrUrGrUrArArUrCrCrCrArU (Antisense)SEQ ID NO: 45 CUR-2079rUrArArGrGrArCrGrCrGrGrArCrUrUrGrUrArCrArCrUrUrCrCrGrGrG (Antisense)SEQ ID NO: 46 CUR-2080rArGrArArArGrArArArGrUrUrCrUrArArCrCrUrGrUrUrCrUrGrUrGrU (Antisense)SEQ ID NO: 47 CUR-2081 +G*+A*+T*T*T*C*A*G*A*G*C*C*G*+C*+A*+GSEQ ID NO: 48 CUR-2082 +G*+A*+C*A*C*A*T*C*C*A*T*C*C*+C*+A*+GSEQ ID NO: 49 CUR-2083 +C*+C*+T*C*G*T*C*A*T*G*T*C*T*+G*+T*+GSEQ ID NO: 50 CUR-0071 C*+T*+T*G*A*A*T*T*G*T*T*T*+G*+T*+A SEQ ID NO: 51CUR-0072 A*+G*+T*T*G*C*A*A*G*A*G*T*+T*+G*+G SEQ ID NO: 52 CUR-0073A*+T*+C*T*G*T*T*C*T*G*C*T*+G*+T*+C SEQ ID NO: 53 CUR-0074C*+A*+T*A*T*T*C*T*T*G*G*A*+C*+G*+A SEQ ID NO: 54 CUR-0075T*+G*+T*G*C*T*G*T*T*G*T*A*+A*+G*+A SEQ ID NO: 55 CUR-0076T*+G*+A*C*A*G*A*G*G*A*G*T*+A*+T*+T

The modulation of a desired target nucleic acid can be carried out inseveral ways known in the art. For example, antisense oligonucleotides,siRNA etc. Enzymatic nucleic acid molecules (e.g., ribozymes) arenucleic acid molecules capable of catalyzing one or more of a variety ofreactions, including the ability to repeatedly cleave other separatenucleic acid molecules in a nucleotide base sequence-specific manner.Such enzymatic nucleic acid molecules can be used, for example, totarget virtually any RNA transcript.

Because of their sequence-specificity, trans-cleaving enzymatic nucleicacid molecules show promise as therapeutic agents for human disease.Enzymatic nucleic acid molecules can be designed to cleave specific RNAtargets within the background of cellular RNA. Such a cleavage eventrenders the mRNA non-functional and abrogates protein expression fromthat RNA. In this manner, synthesis of a protein associated with adisease state can be selectively inhibited.

In general, enzymatic nucleic acids with RNA cleaving activity act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of an enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

Several approaches such as in vitro selection (evolution) strategies(Orgel, (1979) Proc. R. Soc. London, B 205, 435) have been used toevolve new nucleic acid catalysts capable of catalyzing a variety ofreactions, such as cleavage and ligation of phosphodiester linkages andamide linkages.

The development of ribozymes that are optimal for catalytic activitywould contribute significantly to any strategy that employs RNA-cleavingribozymes for the purpose of regulating gene expression. The hammerheadribozyme, for example, functions with a catalytic rate (kcat) of about 1min-1 in the presence of saturating (10 mM) concentrations of Mg2+cofactor. An artificial “RNA ligase” ribozyme has been shown to catalyzethe corresponding self-modification reaction with a rate of about 100min-1. In addition, it is known that certain modified hammerheadribozymes that have substrate binding arms made of DNA catalyze RNAcleavage with multiple turn-over rates that approach 100 min-1. Finally,replacement of a specific residue within the catalytic core of thehammerhead with certain nucleotide analogues gives modified ribozymesthat show as much as a 10-fold improvement in catalytic rate. Thesefindings demonstrate that ribozymes can promote chemical transformationswith catalytic rates that are significantly greater than those displayedin vitro by most natural self-cleaving ribozymes. It is then possiblethat the structures of certain selfcleaving ribozymes may be optimizedto give maximal catalytic activity, or that entirely new RNA motifs canbe made that display significantly faster rates for RNA phosphodiestercleavage.

Intermolecular cleavage of an RNA substrate by an RNA catalyst that fitsthe “hammerhead” model was first shown in 1987 (Uhlenbeck, O. C. (1987)Nature, 328: 596-600). The RNA catalyst was recovered and reacted withmultiple RNA molecules, demonstrating that it was truly catalytic.

Catalytic RNAs designed based on the “hammerhead” motif have been usedto cleave specific target sequences by making appropriate base changesin the catalytic RNA to maintain necessary base pairing with the targetsequences. This has allowed use of the catalytic RNA to cleave specifictarget sequences and indicates that catalytic RNAs designed according tothe “hammerhead” model may possibly cleave specific substrate RNAs invivo.

RNA interference (RNAi) has become a powerful tool for modulating geneexpression in mammals and mammalian cells. This approach requires thedelivery of small interfering RNA (siRNA) either as RNA itself or asDNA, using an expression plasmid or virus and the coding sequence forsmall hairpin RNAs that are processed to siRNAs. This system enablesefficient transport of the pre-siRNAs to the cytoplasm where they areactive and permit the use of regulated and tissue specific promoters forgene expression.

In an embodiment, an oligonucleotide or antisense compound comprises anoligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleicacid (DNA), or a mimetic, chimera, analog or homolog thereof. This termincludes oligonucleotides composed of naturally occurring nucleotides,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftendesired over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

According to the present invention, the oligonucleotides or “antisensecompounds” include antisense oligonucleotides (e.g. RNA, DNA, mimetic,chimera, analog or homolog thereof), ribozymes, external guide sequence(EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, saRNA, aRNA, andother oligomeric compounds which hybridize to at least a portion of thetarget nucleic acid and modulate its function. As such, they may be DNA,RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of oneor more of these. These compounds may be single-stranded,double-stranded, circular or hairpin oligomeric compounds and maycontain structural elements such as internal or terminal bulges,mismatches or loops. Antisense compounds are routinely prepared linearlybut can be joined or otherwise prepared to be circular and/or branched.Antisense compounds can include constructs such as, for example, twostrands hybridized to form a wholly or partially double-strandedcompound or a single strand with sufficient self-complementarity toallow for hybridization and formation of a fully or partiallydouble-stranded compound. The two strands can be linked internallyleaving free 3′ or 5′ termini or can be linked to form a continuoushairpin structure or loop. The hairpin structure may contain an overhangon either the 5′ or 3′ terminus producing an extension of singlestranded character. The double stranded compounds optionally can includeoverhangs on the ends. Further modifications can include conjugategroups attached to one of the termini, selected nucleotide positions,sugar positions or to one of the internucleoside linkages.Alternatively, the two strands can be linked via a non-nucleic acidmoiety or linker group. When formed from only one strand, dsRNA can takethe form of a self-complementary hairpin-type molecule that doubles backon itself to form a duplex. Thus, the dsRNAs can be fully or partiallydouble stranded. Specific modulation of gene expression can be achievedby stable expression of dsRNA hairpins in transgenic cell lines. Whenformed from two strands, or a single strand that takes the form of aself-complementary hairpin-type molecule doubled back on itself to forma duplex, the two strands (or duplex-forming regions of a single strand)are complementary RNA strands that base pair in Watson-Crick fashion.

Once introduced to a system, the compounds of the invention may elicitthe action of one or more enzymes or structural proteins to effectcleavage or other modification of the target nucleic acid or may workvia occupancy-based mechanisms. In general, nucleic acids (includingoligonucleotides) may be described as “DNA-like” (i.e., generally havingone or more 2′-deoxy sugars and, generally, T rather than U bases) or“RNA-like” (i.e., generally having one or more 2′-hydroxyl or2′-modified sugars and, generally U rather than T bases). Nucleic acidhelices can adopt more than one type of structure, most commonly the A-and B-forms. It is believed that, in general, oligonucleotides whichhave B-form-like structure are “DNA-like” and those which haveA-formlike structure are “RNA-like.” In some (chimeric) embodiments, anantisense compound may contain both A- and B-form regions.

The antisense compounds in accordance with this invention can comprisean antisense portion from about 5 to about 80 nucleotides (i.e. fromabout 5 to about 80 linked nucleosides) in length. This refers to thelength of the antisense strand or portion of the antisense compound. Inother words, a single-stranded antisense compound of the inventioncomprises from 5 to about 80 nucleotides, and a double-strandedantisense compound of the invention (such as a dsRNA, for example)comprises a sense and an antisense strand or portion of 5 to about 80nucleotides in length. One of ordinary skill in the art will appreciatethat this comprehends antisense portions of 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides inlength, or any range therewithin.

In one embodiment, the antisense compounds of the invention haveantisense portions of 10 to 50 nucleotides in length. One havingordinary skill in the art will appreciate that this embodiesoligonucleotides having antisense portions of 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides in length, or any range therewithin. In some embodiments,the oligonucleotides are 15 nucleotides in length.

In one embodiment, the antisense or oligonucleotide compounds of theinvention have antisense portions of 12 or 13 to 30 nucleotides inlength. One having ordinary skill in the art will appreciate that thisembodies antisense compounds having antisense portions of 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length, or any range therewithin.

In an embodiment, the oligomeric compounds of the present invention alsoinclude variants in which a different base is present at one or more ofthe nucleotide positions in the compound. For example, if the firstnucleotide is an adenosine, variants may be produced which containthymidine, guanosine or cytidinc at this position. This may be done atany of the positions of the antisense or dsRNA compounds. Thesecompounds are then tested using the methods described herein todetermine their ability to inhibit expression of a target nucleic acid.

In some embodiments, homology, sequence identity or complementarity,between the antisense compound and target is from about 40% to about60%. In some embodiments, homology, sequence identity orcomplementarity, is from about 60% to about 70%. In some embodiments,homology, sequence identity or complementarity, is from about 70% toabout 80%. In some embodiments, homology, sequence identity orcomplementarity, is from about 80% to about 90%. In some embodiments,homology, sequence identity or complementarity, is about 90%, about 92%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100%.

In an embodiment, the antisense oligonucleotides, such as for example,nucleic acid molecules set forth in SEQ ID NOS: 12 to 49 comprise one ormore substitutions or modifications. In one embodiment, the nucleotidesare substituted with locked nucleic acids (LNA).

In an embodiment, the oligonucleotides target one or more regions of thenucleic acid molecules sense and/or antisense of coding and/ornon-coding sequences associated with BDNF and the sequences set forth asSEQ ID NOS: 1 to 11. The oligonucleotides are also targeted tooverlapping regions of SEQ ID NOS: 1 to 11.

Certain preferred oligonucleotides of this invention are chimericoligonucleotides. “Chimeric oligonucleotides” or “chimeras,” in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionof modified nucleotides that confers one or more beneficial properties(such as, for example, increased nuclease resistance, increased uptakeinto cells, increased binding affinity for the target) and a region thatis a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of antisense modulation of gene expression.Consequently, comparable results can often be obtained with shorteroligonucleotides when chimeric oligonucleotides are used, compared tophosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art. In one an embodiment, a chimericoligonucleotide comprises at least one region modified to increasetarget binding affinity, and, usually, a region that acts as a substratefor RNAse H. Affinity of an oligonucleotide for its target (in thiscase, a nucleic acid encoding ras) is routinely determined by measuringthe Tm of an oligonucleotide/target pair, which is the temperature atwhich the oligonucleotide and target dissociate; dissociation isdetected spcctrophotometrically. The higher the Tm, the greater is theaffinity of the oligonucleotide for the target.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotides mimetics as described above.Such; compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In an embodiment, the region of the oligonucleotide which is modifiedcomprises at least one nucleotide modified at the 2′ position of thesugar, most preferably a 2′-Oalkyl, 2′-O-alkyl-O-alkyl or2′-fluoro-modified nucleotide. In other an embodiment, RNA modificationsinclude 2′-fluoro, 2′-amino and 2′ O-methyl modifications on the riboseof pyrimidines, abasic residues or an inverted base at the 3′ end of theRNA. Such modifications are routinely incorporated into oligonucleotidesand these oligonucleotides have been shown to have a higher Tm (i.e.,higher target binding affinity) than; 2′-deoxyoligonucleotides against agiven target. The effect of such increased affinity is to greatlyenhance RNAi oligonucleotide inhibition of gene expression. RNAse H is acellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes;activation of this enzyme therefore results in cleavage of the RNAtarget, and thus can greatly enhance the efficiency of RNAi inhibition.Cleavage of the RNA target can be routinely demonstrated by gelelectrophoresis. In an embodiment, the chimeric oligonucleotide is alsomodified to enhance nuclease resistance. Cells contain a variety of exo-and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance.Oligonucleotides which contain at least one phosphorothioatemodification are presently more preferred. In some cases,oligonucleotide modifications which enhance target binding affinity arealso, independently, able to enhance nuclease resistance.

Specific examples of some preferred oligonucleotides envisioned for thisinvention include those comprising modified backbones, for example,phosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are oligonucleotideswith phosphorothioate backbones and those with heteroatom backbones,particularly CH2-NH—O—CH2, CH,—N(CH3)-O—CH2 [known as amethylene(methylimino) or MMI backbone], CH2-O—N(CH3)-CH2,CH2-N(CH3)-N(CH3)-CH2 and O—N(CH3)-CH2-CH2 backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH). The amide backbonesdisclosed by De Mesmaeker et al. (1995) Ace. Chem. Res. 28:366-374 arealso preferred. Also preferred are oligonucleotides having morpholinobackbone structures (Summerton and Weller, U.S. Pat. No. 5,034,506). Inother an embodiment, such as the peptide nucleic acid (PNA) backbone,the phosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleotides being bound directly or indirectlyto the aza nitrogen atoms of the polyamide backbone. Oligonucleotidesmay also comprise one or more substituted sugar moieties. Preferredoligonucleotides comprise one of the following at the 2′ position: OH,SH, SCH3, F, OCN, OCH3, O(CH2)n CH3, O(CH2)n NH2 or O(CH2)n CH3 where nis from 1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy, substitutedlower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, orN-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2;heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;substituted silyl; an RNA cleaving group; a reporter group; anintercalator; a group for improving the pharmacokinetic properties of anoligonucleotide; or a group for improving the pharmacodynamic propertiesof an oligonucleotide and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy[2′-O—CH2 CH2 OCH3,also known as 2′-O-(2-methoxyethyl)]. Other preferred modificationsinclude 2′-methoxy (2′-O—CH3), 2′-propoxy (2′-OCH2 CH2CH3) and 2′-fluoro(2′-F). Similar modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutyls inplace of the pentofuranosyl group.

Oligonucleotides may also include, additionally or alternatively,nucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleotidesinclude adenine (A), guanine (G), thymine (T), cytosine (C) and uracil(U). Modified nucleotides include nucleotides found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleotides, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and2,6-diaminopurine. A “universal” base known in the art, e.g., inosine,may be included. 5-Mc-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C. and are presently preferredbase substitutions.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety, a cholesteryl moiety, analiphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or apolyethylene glycol chain, or Adamantane acetic acid. Oligonucleotidescomprising lipophilic moieties, and methods for preparing sucholigonucleotides are known in the art, for example, U.S. Pat. Nos.5,138,045, 5,218,105 and 5,459,255.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within an oligonucleotide. The presentinvention also includes oligonucleotides which are chimericoligonucleotides as hereinbefore defined.

In another embodiment, the nucleic acid molecule of the presentinvention is conjugated with another moiety including but not limited toabasic nucleotides, polyether, polyamine, polyamides, peptides,carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in theart will recognize that these molecules can be linked to one or more ofany nucleotides comprising the nucleic acid molecule at severalpositions on the sugar, base or phosphate group.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of one of ordinary skill in the art. It is alsowell known to use similar techniques to prepare other oligonucleotidessuch as the phosphorothioates and alkylated derivatives. It is also wellknown to use similar techniques and commercially available modifiedamidites and controlled-pore glass (CPG) products such as biotin,fluorescein, acridine or psoralen-modified amidites and/or CPG(available from Glen Research, Sterling Va.) to synthesize fluorescentlylabeled, biotinylated or other modified oligonucleotides such ascholesterol-modified oligonucleotides.

In accordance with the invention, use of modifications such as the useof LNA monomers to enhance the potency, specificity and duration ofaction and broaden the routes of administration of oligonucleotidescomprised of current chemistries such as MOE, ANA, FANA, PS etc. Thiscan be achieved by substituting some of the monomers in the currentoligonucleotides by LNA monomers. The LNA modified oligonucleotide mayhave a size similar to the parent compound or may be larger orpreferably smaller. It is preferred that such LNA-modifiedoligonucleotides contain less than about 70%, more preferably less thanabout 60%, most preferably less than about 50% LNA monomers and thattheir sizes are between about 5 and 25 nucleotides, more preferablybetween about 12 and 20 nucleotides.

Preferred modified oligonucleotide backbones comprise, but not limitedto, phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus containing linkages comprise, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides comprise, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds comprise, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen, et al. (1991) Science 254, 1497-1500.

In an embodiment of the invention the oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH2-NH—O—CH2-,-CH2-N (CH3)-O—CH2-known asa methylene (methylimino) or MMI backbone,—CH2-O—N(CH3)-CH2-,-CH2N(CH3)-N(CH3)CH2-and-O—N(CH3)-CH2-CH2- whereinthe native phosphodiester backbone is represented as —O—P—O—CH2- of theabove referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove referenced U.S. Pat. No. 5,602,240. Also preferred areoligonucleotides having morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C to CO alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O (CH2)n OmCH3, O(CH2)n,OCH3,O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON(CH3)2 where n and mcan be from 1 to about 10. Other preferred oligonucleotides comprise oneof the following at the 2′ position: C to CO, (lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN,Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification comprises 2′-methoxyethoxy (2′-O—CH2CH2OCH3,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) i.e., an alkoxyalkoxygroup. A further preferred modification comprises2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH2)2.

Other preferred modifications comprise 2′-methoxy (2′-O CH3),2′-aminopropoxy (2′-O CH2CH2CH2NH2) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures comprise, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514, 785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646, 265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference.

Oligonucleotides may also comprise nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleotides comprise the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleotides comprise other synthetic andnatural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleotides comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., ‘AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, ‘Antisense Research and Applications’,pages 289-302, Crooke, S. T. and Lebleu, B. ca., CRC Press, 1993.Certain of these nucleotides are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Thesecomprise 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and0-6 substituted purines, comprising 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Researchand Applications’, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-Omethoxyethyl sugar modifications.

Representative United States patents that teach the preparation of theabove noted modified nucleotides as well as other modified nucleotidescomprise, but are not limited to, U.S. Pat. No. 3,687,808, as well asU.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941,each of which is herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates, which enhance the activity, cellular distribution, orcellular uptake of the oligonucleotide.

Such moieties comprise but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or Adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-toxycholesterol moiety.

Representative United States patents that teach the preparation of sucholigonucleotides conjugates comprise, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486, 603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

Drug Discovery:

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between Brain derived neurotrophic factor (BDNF)polynucleotides and a disease state, phenotype, or condition. Thesemethods include detecting or modulating BDNF polynucleotides comprisingcontacting a sample, tissue, cell, or organism with the compounds of thepresent invention, measuring the nucleic acid or protein level of BDNFpolynucleotides and/or a related phenotypic or chemical endpoint at sometime after treatment, and optionally comparing the measured value to anon-treated sample or sample treated with a further compound of theinvention. These methods can also be performed in parallel or incombination with other experiments to determine the function of unknowngenes for the process of target validation or to determine the validityof a particular gene product as a target for treatment or prevention ofa particular disease, condition, or phenotype.

Assessing Up-Regulation or Inhibition of Gene Expression:

Transfer of an exogenous nucleic acid into a host cell or organism canbe assessed by directly detecting the presence of the nucleic acid inthe cell or organism. Such detection can be achieved by several methodswell known in the art. For example, the presence of the exogenousnucleic acid can be detected by Southern blot or by a polymerase chainreaction (PCR) technique using primers that specifically amplifynucleotide sequences associated with the nucleic acid. Expression of theexogenous nucleic acids can also be measured using conventional methodsincluding gene expression analysis. For instance, mRNA produced from anexogenous nucleic acid can be detected and quantified using a Northernblot and reverse transcription PCR (RT-PCR).

Expression of RNA from the exogenous nucleic acid can also be detectedby measuring an enzymatic activity or a reporter protein activity. Forexample, antisense modulatory activity can be measured indirectly as adecrease or increase in target nucleic acid expression as an indicationthat the exogenous nucleic acid is producing the effector RNA. Based onsequence conservation, primers can be designed and used to amplifycoding regions of the target genes. Initially, the most highly expressedcoding region from each gene can be used to build a model control gene,although any coding or non coding region can be used. Each control geneis assembled by inserting each coding region between a reporter codingregion and its poly(A) signal. These plasmids would produce an mRNA witha reporter gene in the upstream portion of the gene and a potential RNAitarget in the 3′ non-coding region. The effectiveness of individualantisense oligonucleotides would be assayed by modulation of thereporter gene. Reporter genes useful in the methods of the presentinvention include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP),cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase(Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivativesthereof. Multiple selectable markers are available that conferresistance to ampicillin, bleomycin, chloramphenicol, gentamycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, and tetracycline. Methods to determine modulation of areporter gene are well known in the art, and include, but are notlimited to, fluorometric methods (e.g. fluorescence spectroscopy,Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy),antibiotic resistance determination.

BDNF protein and mRNA expression can be assayed using methods known tothose of skill in the art and described elsewhere herein. For example,immunoassays such as the ELISA can be used to measure protein levels.BDNF ELISA assay kits are available commercially, e.g., from R&D Systems(Minneapolis, Minn.).

In embodiments, BDNF expression (e.g., mRNA or protein) in a sample(e.g., cells or tissues in vivo or in vitro) treated using an antisenseoligonucleotide of the invention is evaluated by comparison with BDNFexpression in a control sample. For example, expression of the proteinor nucleic acid can be compared using methods known to those of skill inthe art with that in a mock-treated or untreated sample. Alternatively,comparison with a sample treated with a control antisenseoligonucleotide (e.g., one having an altered or different sequence) canbe made depending on the information desired. In another embodiment, adifference in the expression of the BDNF protein or nucleic acid in atreated vs. an untreated sample can be compared with the difference inexpression of a different nucleic acid (including any standard deemedappropriate by the researcher, e.g., a housekeeping gene) in a treatedsample vs. an untreated sample.

Observed differences can be expressed as desired, e.g., in the form of aratio or fraction, for use in a comparison with control. In embodiments,the level of BDNF mRNA or protein, in a sample treated with an antisenseoligonucleotide of the present invention, is increased or decreased byabout 1.25-fold to about 10-fold or more relative to an untreated sampleor a sample treated with a control nucleic acid. In embodiments, thelevel of BDNF mRNA or protein is increased or decreased by at leastabout 1.25-fold, at least about 1.3-fold, at least about 1.4-fold, atleast about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold,at least about 1.8-fold, at least about 2-fold, at least about 2.5-fold,at least about 3-fold, at least about 3.5-fold, at least about 4-fold,at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold,at least about 6-fold, at least about 6.5-fold, at least about 7-fold,at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold,at least about 9-fold, at least about 9.5-fold, or at least about10-fold or more.

Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention can be utilized for diagnostics,therapeutics, and prophylaxis, and as research reagents and componentsof kits. Furthermore, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes orto distinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics and in various biological systems, thecompounds of the present invention, either alone or in combination withother compounds or therapeutics, are useful as tools in differentialand/or combinatorial analyses to elucidate expression patterns of aportion or the entire complement of genes expressed within cells andtissues.

As used herein the term “biological system” or “system” is defined asany organism, cell, cell culture or tissue that expresses, or is madecompetent to express products of the Brain derived neurotrophic factor(BDNF) genes. These include, but are not limited to, humans, transgenicanimals, cells, cell cultures, tissues, xenografts, transplants andcombinations thereof.

As one non limiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundsthat affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays, SAGE (serial analysis of gene expression),READS (restriction enzyme amplification of digested cDNAs), TOGA (totalgene expression analysis), protein arrays and proteomics, expressedsequence tag (EST) sequencing, subtractive RNA fingerprinting (SuRF),subtractive cloning, differential display (DD), comparative genomichybridization, FISH (fluorescent in situ hybridization) techniques andmass spectrometry methods.

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encoding Brainderived neurotrophic factor (BDNF). For example, oligonucleotides thathybridize with such efficiency and under such conditions as disclosedherein as to be effective BDNF modulators are effective primers orprobes under conditions favoring gene amplification or detection,respectively. These primers and probes are useful in methods requiringthe specific detection of nucleic acid molecules encoding BDNF and inthe amplification of said nucleic acid molecules for detection or foruse in further studies of BDNF. Hybridization of the antisenseoligonucleotides, particularly the primers and probes, of the inventionwith a nucleic acid encoding BDNF can be detected by means known in theart. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabeling of the oligonucleotide, or any othersuitable detection means. Kits using such detection means for detectingthe level of BDNF in a sample may also be prepared.

The specificity and sensitivity of antisense are also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs have beensafely and effectively administered to humans and numerous clinicaltrials are presently underway. It is thus established that antisensecompounds can be useful therapeutic modalities that can be configured tobe useful in treatment regimes for the treatment of cells, tissues andanimals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofBDNF polynucleotides is treated by administering antisense compounds inaccordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to the animalin need of treatment, a therapeutically effective amount of BDNFmodulator. The BDNF modulators of the present invention effectivelymodulate the activity of the BDNF or modulate the expression of the BDNFprotein. In one embodiment, the activity or expression of BDNF in ananimal is inhibited by about 10% as compared to a control. Preferably,the activity or expression of BDNF in an animal is inhibited by about30%. More preferably, the activity or expression of BDNF in an animal isinhibited by 50% or more. Thus, the oligomeric compounds modulateexpression of Brain derived neurotrophic factor (BDNF) mRNA by at least10%, by at least 50%, by at least 25%, by at least 30%, by at least 40%,by at least 50%, by at least 60%, by at least 70%, by at least 75%, byat least 80%, by at least 85%, by at least 90%, by at least 95%, by atleast 98%, by at least 99%, or by 100% as compared to a control.

In one embodiment, the activity or expression of Brain derivedneurotrophic factor (BDNF) in an animal is increased by about 10% ascompared to a control. Preferably, the activity or expression of BDNF inan animal is increased by about 30%. More preferably, the activity orexpression of BDNF in an animal is increased by 50% or more. Thus, theoligomeric compounds modulate expression of BDNF mRNA by at least 10%,by at least 50%, by at least 25%, by at least 30%, by at least 40%, byat least 50%, by at least 60%, by at least 70%, by at least 75%, by atleast 80%, by at least 85%, by at least 90%, by at least 95%, by atleast 98%, by at least 99%, or by 100% as compared to a control.

For example, the increase or reduction of the expression of Brainderived neurotrophic factor (BDNF) may be measured in serum, blood,adipose tissue, liver or any other body fluid, tissue or organ of theanimal. Preferably, the cells contained within said fluids, tissues ororgans being analyzed contain a nucleic acid molecule encoding BDNFpeptides and/or the BDNF protein itself.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

Conjugates

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92/09196, filed Oct. 23,1992, and U.S. Pat. No. 6,287,860, which are incorporated herein byreference. Conjugate moieties include, but are not limited to, lipidmoieties such as a cholesterol moiety, cholic acid, a thioether, e.g.,hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate, a polyamine or apolyethylene glycol chain, or Adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

Representative United States patents that teach the preparation of sucholigonucleotides conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,165; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

Although, the antisense oligonucleotides do not need to be administeredin the context of a vector in order to modulate a target expressionand/or function, embodiments of the invention relates to expressionvector constructs for the expression of antisense oligonucleotides,comprising promoters, hybrid promoter gene sequences and possess astrong constitutive promoter activity, or a promoter activity which canbe induced in the desired case.

In an embodiment, invention practice involves administering at least oneof the foregoing antisense oligonucleotides with a suitable nucleic aciddelivery system. In one embodiment, that system includes a non-viralvector operably linked to the polynucleotide. Examples of such nonviralvectors include the oligonucleotide alone (e.g. any one or more of SEQID NOS: 12 to 49) or in combination with a suitable protein,polysaccharide or lipid formulation.

Additionally suitable nucleic acid delivery systems include viralvector, typically sequence from at least one of an adenovirus,adenovirus-associated virus (AAV), helper-dependent adenovirus,retrovirus, or hemagglutinatin virus of Japan-liposome (HVJ) complex.Preferably, the viral vector comprises a strong eukaryotic promoteroperably linked to the polynucleotide e.g., a cytomegalovirus (CMV)promoter.

Additionally preferred vectors include viral vectors, fusion proteinsand chemical conjugates. Retroviral vectors include Moloney murineleukemia viruses and HIV-based viruses. One preferred H1V-based viralvector comprises at least two vectors wherein the gag and pol genes arefrom an HIV genome and the env gene is from another virus. DNA viralvectors are preferred. These vectors include pox vectors such asorthopox or avipox vectors, herpesvirus vectors such as a herpes simplexI virus (HSV) vector, Adenovirus Vectors and Adeno-associated VirusVectors.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein by reference.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration.

For treating tissues in the central nervous system, administration canbe made by, e.g., injection or infusion into the cerebrospinal fluid.Administration of antisense RNA into cerebrospinal fluid is described,e.g., in U.S. Pat. App. Pub. No. 2007/0117772, “Methods for slowingfamilial ALS disease progression,” incorporated herein by reference inits entirety.

When it is intended that the antisense oligonucleotide of the presentinvention be administered to cells in the central nervous system,administration can be with one or more agents capable of promotingpenetration of the subject antisense oligonucleotide across theblood-brain barrier. Injection can be made, e.g., in the entorhinalcortex or hippocampus. Delivery of neurotrophic factors byadministration of an adenovirus vector to motor neurons in muscle tissueis described in, e.g., U.S. Pat. No. 6,632,427,“Adenoviral-vector-mediated gene transfer into medullary motor neurons,”incorporated herein by reference. Delivery of vectors directly to thebrain, e.g., the striatum, the thalamus, the hippocampus, or thesubstantia nigra, is known in the art and described, e.g., in U.S. Pat.No. 6,756,523, “Adenovirus vectors for the transfer of foreign genesinto cells of the central nervous system particularly in brain,”incorporated herein by reference. Administration can be rapid as byinjection or made over a period of time as by slow infusion oradministration of slow release formulations.

The subject antisense oligonucleotides can also be linked or conjugatedwith agents that provide desirable pharmaceutical or pharmacodynamicproperties. For example, the antisense oligonucleotide can be coupled toany substance, known in the art to promote penetration or transportacross the blood-brain barrier, such as an antibody to the transferrinreceptor, and administered by intravenous injection. The antisensecompound can be linked with a viral vector, for example, that makes theantisense compound more effective and/or increases the transport of theantisense compound across the blood-brain barrier. Osmotic blood brainbarrier disruption can also be accomplished by, e.g., infusion of sugarsincluding, but not limited to, meso erythritol, xylitol, D(+) galactose,D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(−) fructose, D(−)mannitol, D(+) glucose, D(+) arabinose, D(−) arabinose, cellobiose, D(+)maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(−) ribose,adonitol, D(+) arabitol, L(−) arabitol, D(+) fucose, L(−) fucose, D(−)lyxose, L(+) lyxose, and L(−) lyxose, or amino acids including, but notlimited to, glutamine, lysine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glycine, histidine, leucine, methionine,phenylalanine, proline, serine, threonine, tyrosine, valine, andtaurine. Methods and materials for enhancing blood brain barrierpenetration are described, e.g., in U.S. Pat. No. 4,866,042, “Method forthe delivery of genetic material across the blood brain barrier,” U.S.Pat. No. 6,294,520, “Material for passage through the blood-brainbarrier,” and U.S. Pat. No. 6,936,589, “Parenteral delivery systems,”all incorporated herein by reference in their entirety.

The subject antisense compounds may be admixed, encapsulated, conjugatedor otherwise associated with other molecules, molecule structures ormixtures of compounds, for example, liposomes, receptor-targetedmolecules, oral, rectal, topical or other formulations, for assisting inuptake, distribution and/or absorption. For example, cationic lipids maybe included in the formulation to facilitate oligonucleotide uptake. Onesuch composition shown to facilitate uptake is LIPOFECTIN (availablefrom GIBCO-BRL, Bethesda, Md.).

Oligonucleotides with at least one 2′-O-methoxyethyl modification arebelieved to be particularly useful for oral administration.Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogeneous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug that may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes that are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids. When incorporated into liposomes, these specialized lipidsresult in liposomes with enhanced circulation lifetimes relative toliposomes lacking such specialized lipids. Examples of stericallystabilized liposomes are those in which part of the vesicle-forminglipid portion of the liposome comprises one or more glycolipids or isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. Liposomes and their uses are furtherdescribed in U.S. Pat. No. 6,287,860.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein by reference.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating nonsurfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein by reference.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoyl-phosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoyl-phosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein by reference. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein by reference.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents that function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bischloroethyl-nitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. For example, the first targetmay be a particular antisense sequence of Brain derived neurotrophicfactor (BDNF), and the second target may be a region from anothernucleotide sequence. Alternatively, compositions of the invention maycontain two or more antisense compounds targeted to different regions ofthe same Brain derived neurotrophic factor (BDNF) nucleic acid target.Numerous examples of antisense compounds are illustrated herein andothers may be selected from among suitable compounds known in the art.Two or more combined compounds may be used together or sequentially.

Dosing:

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient.

Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC50s found to be effective in vitro andin vivo animal models. In general, dosage is from 0.01 μg to 10 mg perkg of body weight, and may be given once or more daily, weekly, monthlyor yearly, or even once every 2 to 20 years. Persons of ordinary skillin the art can easily estimate repetition rates for dosing based onmeasured residence times and concentrations of the drug in bodily fluidsor tissues. Following successful treatment, it may be desirable to havethe patient undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the oligonucleotide is administered inmaintenance doses, ranging from 0.01 μg to 10 mg per kg of body weight,once or more daily, to once every 2-20 years.

In embodiments, a patient is treated with a dosage of drug that is atleast about 1, at least about 2, at least about 3, at least about 4, atleast about 5, at least about 6, at least about 7, at least about 8, atleast about 9, at least about 10, at least about 15, at least about 20,at least about 25, at least about 30, at least about 35, at least about40, at least about 45, at least about 50, at least about 60, at leastabout 70, at least about 80, at least about 90, or at least about 10mg/kg body weight. Certain injected dosages of antisenseoligonucleotides are described, e.g., in U.S. Pat. No. 7,563,884,“Antisense modulation of PTP1B expression,” incorporated herein byreference in its entirety.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Numerous changes to the disclosedembodiments can be made in accordance with the disclosure herein withoutdeparting from the spirit or scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described embodiments.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document, Applicants donot admit any particular reference is “prior art” to their invention.Embodiments of inventive compositions and methods are illustrated in thefollowing examples.

EXAMPLES

The following non-limiting Examples serve to illustrate selectedembodiments of the invention. It will be appreciated that variations inproportions and alternatives in elements of the components shown will beapparent to those skilled in the art and are within the scope ofembodiments of the present invention.

Example 1 Design of Antisense Oligonucleotides Specific for a NucleicAcid Molecule Antisense to a Brain Derived Neurotrophic Factor (BDNF)and/or a Sense Strand of BDNF Polynucleotide

As indicated above the term “oligonucleotide specific for” or“oligonucleotide targets” refers to an oligonucleotide having a sequence(i) capable of forming a stable complex with a portion of the targetedgene, or (ii) capable of forming a stable duplex with a portion of anmRNA transcript of the targeted gene.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs (e.g. IDT AntiSense Design, IDT OligoAnalyzer) thatautomatically identify in each given sequence subsequences of 19-25nucleotides that will form hybrids with a target polynucleotide sequencewith a desired melting temperature (usually 50-60° C.) and will not formself-dimers or other complex secondary structures.

Selection of appropriate oligonucleotides is further facilitated byusing computer programs that automatically align nucleic acid sequencesand indicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of genes and intergenic regions of agiven genome allows the selection of nucleic acid sequences that displayan appropriate degree of specificity to the gene of interest. Theseprocedures allow the selection of oligonucleotides that exhibit a highdegree of complementarity to target nucleic acid sequences and a lowerdegree of complementarity to other nucleic acid sequences in a givengenome. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays.

The hybridization properties of the oligonucleotides described hereincan be determined by one or more in vitro assays as known in the art.For example, the properties of the oligonucleotides described herein canbe obtained by determination of binding strength between the targetnatural antisense and a potential drug molecules using melting curveassay.

The binding strength between the target natural antisense and apotential drug molecule (Molecule) can be estimated using any of theestablished methods of measuring the strength of intermolecularinteractions, for example, a melting curve assay.

Melting curve assay determines the temperature at which a rapidtransition from double-stranded to single-stranded conformation occursfor the natural antisense/Molecule complex. This temperature is widelyaccepted as a reliable measure of the interaction strength between thetwo molecules.

A melting curve assay can be performed using a cDNA copy of the actualnatural antisense RNA molecule or a synthetic DNA or RNA nucleotidecorresponding to the binding site of the Molecule. Multiple kitscontaining all necessary reagents to perform this assay are available(e.g. Applied Biosystems Inc. MeltDoctor kit). These kits include asuitable buffer solution containing one of the double strand DNA (dsDNA)binding dyes (such as ABI HRM dyes, SYBR Green, SYTO, etc.). Theproperties of the dsDNA dyes are such that they emit almost nofluorescence in free form, but are highly fluorescent when bound todsDNA.

To perform the assay the cDNA or a corresponding oligonucleotide aremixed with Molecule in concentrations defined by the particularmanufacturer's protocols. The mixture is heated to 95° C. to dissociateall pre-formed dsDNA complexes, then slowly cooled to room temperatureor other lower temperature defined by the kit manufacturer to allow theDNA molecules to anneal. The newly formed complexes are then slowlyheated to 95° C. with simultaneous continuous collection of data on theamount of fluorescence that is produced by the reaction. Thefluorescence intensity is inversely proportional to the amounts of dsDNApresent in the reaction. The data can be collected using a real time PCRinstrument compatible with the kit (e.g. ABI's StepOne Plus Real TimePCR System or lightTyper instrument, Roche Diagnostics, Lewes, UK).

Melting peaks are constructed by plotting the negative derivative offluorescence with respect to temperature (−d(Fluorescence)/dT) on they-axis) against temperature (x-axis) using appropriate software (forexample lightTyper (Roche) or SDS Dissociation Curve, ABI). The data isanalyzed to identify the temperature of the rapid transition from dsDNAcomplex to single strand molecules. This temperature is called Tm and isdirectly proportional to the strength of interaction between the twomolecules. Typically, Tm will exceed 40° C.

Design of Modified AntagoNAT Molecules:

A number of DNA based antisense oligonucleotides were designed andtested, termed AntagoNATs, targeting noncoding Bdnf-AS and otherantisense transcripts. Various AntagoNATs were designed ranging from 12to 20 nucleotides in length with or without full phosphorothioatemodification plus/minus 2-O′-methyl RNA or LNA modified nucleotides.highest efficacy was observed on Bdnf mRNA level with 16-nucleotidephosphorothioate gapmer with three LNA-modified nucleotides at each end(XXXnnnnnnnnnnXXX). For blocking interactions between human BDNFsense-antisense transcripts, 14-nucleotide mixmers containing both LNAand 2-O′-methyl RNA molecules were used. Although these 2-O′-methylRNA-modified oligonucleotides are suggested to only block the RNA,marginal downregulation of targeted RNAs was observed in this experiment(FIG. 11). Sequences of various AntagoNATs, as well as all other siRNAs,primers and probes used for these studies are listed in Table 1.

Example 2 Modulation of BDNF Polynucleotides

All antisense oligonucleotides used in Example 2 were designed asdescribed in Example 1. The manufacturer (IDT Inc. of Coralville, Iowa)was instructed to manufacture the designed phosphothioate bondoligonucleotides and provided the designed phosphothioate analogs shownin Table 1. The asterisk designation between nucleotides indicates thepresence of phosphothioate bond. The oligonucleotides required for theexperiment in Example 2 can be synthesized using any appropriate stateof the art method, for example the method used by IDT: on solid support,such as a 5 micron controlled pore glass bead (CPG), usingphosphoramidite monomers (normal nucleotides with all active groupsprotected with protection groups, e.g. trityl group on sugar, benzoyl onA and C and N-2-isobutyryl on G). Protection groups prevent the unwantedreactions during oligonucleotide synthesis. Protection groups areremoved at the end of the synthesis process. The initial nucleotide islinked to the solid support through the 3′carbon and the synthesisproceeds in the 3′ to 5′direction. The addition of a new base to agrowing oligonucleotide chain takes place in four steps: 1) theprotection group is removed from the 5′ oxygen of the immobilizednucleotide using trichloroacetic acid; 2) the immobilized and thenext-in-sequence nucleotides are coupled together using tetrazole; thereaction proceeds through a tetrazolyl phosphoramidite intermediate; 3)the unreacted free nucleotides and reaction byproducts are washed awayand the unreacted immobilized oligonucleotides are capped to preventtheir participation in the next round of synthesis; capping is achievedby acetylating the free 5′ hydroxyl using acetic anhydride and N-methylimidazole; 4) to stabilize the bond between the nucleotides thephosphorus is oxidized using iodine and water, if a phosphodiester bondis to be produced, or Beaucage reagent(3H-1,2-benzodithiol-3-one-1,1-dioxide), if a phosphothioate bond isdesired. By alternating the two oxidizing agents, a chimeric backbonecan be constructed. The four step cycle described above is repeated forevery nucleotide in the sequence. When the complete sequence issynthesized, the oligonucleotide is cleaved from the solid support anddeprotected using ammonium hydroxide at high temperature. Protectiongroups are washed away by desalting and the remaining oligonucleotidesare lyophilized.

Treatment of Hek293 Cells with Different siRNA to Quantify the Amount ofBDNF mRNA

1. Hek293 cells from ATCC (cat#CRL-1573) were grown in MEM/EBSS (Hyclonecat #SH30024)+10% FBS+penicillin+streptomycin at 37° C. and 5% CO₂. Oneday before the experiment the cells were replated at the density of5×10⁵/well into 6 well plates and incubated at 37° C. and 5% CO₂.

2. On the day of the experiment the media in the 6 well plates waschanged to fresh MEM/EBSS+10% FBS.

3. All BDNF-AntagoNAT (oligonucleotide antisense of BDNF-AS) werediluted to the concentration of 20 uM and the BDNF-AS siRNA (siRNAcomplementary of BDNF-AS at 10 uM; both oligonucleotide compounds aremanufactured by IDT. To dose one well, 2 μl of this solution wasincubated with 400 μl of Opti-MEM media (Gibco cat#31985-070) and 4 ulof Lipofectamine 2000 (Invitrogen cat#11668019) at room temperature for20 min and applied drop wise to one well of the 6 well plates with HepG2cells. Similar mixture including 2 μl of water instead of theoligonucleotide solution was used for the mock-transfected controls.

4. After 3-18 h of incubation at 37° C. and 5% CO₂ the media was changedto fresh MEM/EBSS+10% FBS+penicillin+streptomycin.

5. 48 h after addition of antisense oligonuclotides was performed. Themedia was then removed and RNA was extracted from the cells using SVTotal RNA Isolation System from Promega (cat #Z3105) or RNeasy Total RNAIsolation kit from Qiagen (cat#74181) following the manufacturers'instructions.

6. 200-400 ng of extracted RNA was added to the reverse transcriptionreaction performed using random hexamers, 2.5 mM mixture of dNTP, MgCl₂and appropriate buffer. The cDNA (20-40 ng) from this reversetranscription reaction was used to monitor gene expression by real timePCR using ABI Taqman Gene Expression Mix (cat#4369510) and 300 nM offorward and reverse primers, and 200 nM of probe in a final reactionvolume of 15 μl. The primers/probes were designed using FileBuildersoftware (Applied Biosystem). Primers were strand specific forsense-antisense pairs and the probes covered exon boundaries toeliminate the chance of genomic DNA amplification. The ABI assay forhuman BDNF was Applied Biosystems Taqman Gene Expression Assay:Hs00542425_s1 (BDNF) by Applied Biosystems Inc., Foster City Calif.).The following PCR cycle was used: 50° C. for 2 min, 95° C. for 10 min,40 cycles of (95° C. for 15 seconds, 60° C. for 1 min) using GeneAmp7900 Machine (Applied Biosystems). Fold change in gene expression aftertreatment with antisense oligonucleotides was calculated based on thedifference in 18S-normalized dCt values between treated andmock-transfected samples.

7. Detection oligos for BDNF-AS:

ABI assay ID Hs00417345_m1 (SEQ ID No.: 65) Context sequenceGCACACCTGGAGATACTCTATTATA

8. Detection oligos for BDNF:

ABI assay ID Hs00542425_s1 (SEQ ID No.: 66) CCTGCAGAATGGCCTGGAATTACAADetection oligos for BDNF-AS: ABI assay ID Hs00417345_m1(SEQ ID No.: 65) Context sequence GCACACCTGGAGATACTCTATTATADetection oligos for BDNF: ABI assay ID Hs00542425_s1 (SEQ ID No.: 66)CCTGCAGAATGGCCTGGAATTACAA

9. The results are based on cycle threshold (Ct) values. The calculateddifferences between the Ct values for experimental and references genes(18S RNA) as ddCt and graphed as a percentage of each RNA to calibratorsample.

Results:

Transfection of several human and mouse cells lines including HEK293Tcells with different siRNA that target non-overlapping regions of theBDNF-AS transcript show a 2-6 fold upregulation of the BDNF transcript(FIG. 1 a and FIG. 6) at 48 h. The up-regulation of BDNF was not relatedto the choice of endogenous controls (FIG. 5 a-b). The up-regulation ofdid not affect the regulation of other BDNF neighboring genes (FIG. 9).

FIG. 5 shows BDNF-AS knockdown leads to BDNF mRNA upregulation.

Knockdown of BDNF-AS, using siRNAs-1 (10 nM) targeting thenon-overlapping region of the BDNF-AS transcript, caused a 6-foldupregulation of BDNF (sense) mRNA (****=P<0.0001). Results depicted herewere obtained from experiments in HEK293T cells, using beta actin (leftpanel) or 18S rRNA (right panel) as endogenous controls and the mocktransfection as a reference sample. This experiment is intended to showthat choice of endogenous controls or reference calibrator sample doesnot change the observed upregulation of BDNF mRNA.

FIG. 6 shows Posttranscriptional regulation of Bdnf expression.Transfected N2a cells with combination of mBdnf-AntagoNAT9 targetingmouse Bdnf-AS transcript and Drosha siRNA targeting Drosha protein,which is involved in microRNA (miRNA) processing. Bdnf mRNA upregulationwas observed following treatment of cells with mBdnf-ANtagoNAT9 (***=pvalue <0.0001). Addition of Drosha siRNA marginally increased Bdnftranscript over mBdnf-AntagoNAT9 treatment (*=p value <0.05). Thisexperiment may suggest involvement of other post-transcriptionalmechanisms, such as miRNAs in regulation of Bdnf transcript.

FIG. 9 shows BDNF-AS knockdown neither changes the level of TrkB norBDNF neighboring genes (Let7C and KIF18A) in both directions: LIN7C andKIF18A are genes located 3′ downstream and 5′ upstream of BDNF,respectively. Neurotrophic tyrosine kinase, receptor, type 2 (TrkB)encodes a membrane-bound receptor for BDNF and is located on a differentchromosome (Chr-9) as BDNF. It was determined whether these genes werealtered upon depletion of the BDNF-AS transcript. HEK293T cells weretransfected with control siRNA or BDNF-AS siRNA and measured severaltranscript levels. It was observed that the BDNF-AS transcript wasdownregulated and that BDNF mRNA was upregulated as indicated elsewherein this manuscript. It was found that the knockdown of BDNFAS has noeffect on TrkB expression or on the neighboring genes Let7C and KIF18A.These data suggest that upon BDNF-AS depletion, there is alocus-specific alteration of BDNF expression.

Treatment of Hek293 Cells with One siRNA in a Time Course of 0-96 h toQuantify the Amount of BDNF and BDNF-AS

The methodology followed was same as in Treatment of Hek293 cells withsiRNA but this time the cells are harvested at 0 to 96 h after additionof the oligos.

Results:

The time course of BDNF and BDNF-AS expression shows an optimumup-regulation of BDNF due to the siRNA at 48 h simultaneously to anoptimum downregulation of the BDNF-AS (FIG. 1 b).

Treatment of Hek293 Cells with Different hBDNF-AntagoNATs to Quantifythe Amount of BDNF and BDNF-AS

The methodology followed was same as in Treatment of Hek293 cells withsiRNA but this time the cells are treated with AntagoNATs.

Results:

The BDNF-AS transcript contains a 225-nucleotide overlapping region thathas full complementarity to the BDNF mRNA. The RNA-RNA interactions maybe responsible for the discordant regulation of BDNF by its antisensetranscript. To determine the regulatory role of BDNF-AS on BDNF mRNA,the gapmers (AntagoNATs) containing both LNA and 2′OMe RNA modificationswere utilized to block the interaction between sense and antisensetranscripts. The overlapping region was covered by tilinghBDNF-AatagoNATs. It was found that the use of hBDNF-AntagoNATsupregulates the BDNF mRNA. A marginal downregulation of BDNF-AStranscript was observed, which was not expected for 2′OMe-RNA containingblocking oligos. The 16 hBDNF-AntagoNATs were tested and it was foundthat blocking the first half of the BDNF-AS overlapping region has agreater effect on the upregulation of BDNF mRNA. Specifically,hBDNF-AntagoNAT1 and hBDNFAntagoNAT4 caused significant upregulation ofBDNF mRNA. Unlike synthetic siRNAs, antisense oligonucleotides aresingle-stranded and can be shorter in length; therefore, reducingnon-specific (off-target) binding effects. Single-stranded lockednucleic acid (LNA)-modified oligonucleotides are generally moreeffective, in vivo, compared to unmodified siRNAs (FIG. 7).

Treatment of Mouse N2a Cells with Different mBDNF-AntagoNATs to Quantifythe Amount of BDNF and BDNF-AS

The methodology followed was same as in Treatment of Hek293 cells withdifferent hBDNF-AntagoNATs to quantify the amount of BDNF and BDNF-ASbut this time the cells are N2a cells. Furthermore, the following PCRcycle was used: 50° C. for 2 min, 95° C. for 10 min, 50 cycles of (95°C. for 15 seconds, 60° C. for 1 min) using GeneAmp 7900 Machine (AppliedBiosystems).

Results FIG. 8 shows Inhibition of the mouse Bdnf-AS transcript in N2acells, by AntagoNATs: that blocking of the overlap region between humanBDNF sense and antisense transcripts upregulates BDNF mRNA levels. Itwas then determined if a similar regulatory mechanism exists in a mousecell line and tested 11 mBdnf-AntagoNATs that target the mouse Bdnf-AStranscript. mBdnf-AntagoNATs contain a phosphorothioate backbone andthree LNA-modified nucleotides at both 3′ and 5′ ends. Controloligonucleotides have a similar backbone and modifications, but do nottarget any sequence in the mammalian genomes. Two mBdnf-AntagoNATs(mBdnf-AntagoNA3 and mBdnf-AntagoNAT-9) were able to increase Bdnf mRNAlevels in N2a cells. In sum, blocking the mouse Bdnf-AS transcript withsingle-stranded AntagoNATs (16-mer) caused an upregulation of Bdnf mRNAlevels in mouse N2a cells. These data suggest that the antisensetranscript of Bdnf exerts a suppressive effect on Bdnf mRNA.

Treatment of Hek293 Cells with Different siRNA to Quantify the BDNFProtein

The methodology followed was same as in Treatment of Hek293 cells withdifferent siRNA to quantify the amount of BDNF mRNA, except at step 5where, 48 h after addition of siRNA was performed. The media was thenremoved and cells were disrupted and their levels of BDNF protein wasquantified by ELISA (FIG. 1 c) and western blot (FIG. 1 d).

Western Blot:

HEK293T cells were transfected with 10 nM of BDNF-AS, or control siRNA.The cells were disrupted, 48 h post transfection, with 200 μl of Laemmlisample buffer (Biorad) containing 350 mM DTT. 20 μl of the lysate wasseparated on a 10% SDS PAGE and transferred it to a nitrocellulosemembrane overnight. Then the incubated the membrane with primaryantibody for MecP2 (Abcam), BDNF (Promega, catalog number G164B) andsecondary antibody conjugated to HRP. After addition of HRP substrate,the chemiluminescent signal was detected with X-ray film. The samemembrane was stripped and reused it for detection of P-Actin as aloading control.

ELISA:

Cells were transfected with 20 nM of BDNF-AS siRNA or control siRNA. Thecell supernatant was collected for ELISA experiments. Alternatively,total protein was extracted from mouse brain tissues embedded in proteinextraction buffer plus protease inhibitors (BCA kit, Fisher) andhomogenized with the bioruptor and metal beads. Total protein wasmeasured using BCA protein assay kit (Pierce catalog number 23227) andsample loads were normalized to total protein concentrations. the ELISAkits were purchased for human BDNF from Promega (catalog number G7611)or mouse Bdnf Millipore (catalog number CYT306) and ELISA was performedfollowing the supplier's protocol. Average absorbance was subtracted ofthree repeats at 450 nm from background and normalized it to the controlsample.

Treatment of Hek293 Cells (not Sure) with Different Concentrations ofmBDNF-AntagoNAT9 to Quantify the BDNF mRNA

The methodology followed was same as in Treatment of Hek293 cells withdifferent siRNA to quantify the amount of BDNF mRNA except at step 3,where all mBDNF-AntagoNAT9 was diluted to different concentration suchas the final of 11 different concentrations were applied to the cells(1:3 serial dilutions ranging from 300 nM to 5 pM) using the sameproportional amounts of Lipofectamine 2000 (Invitrogen cat#11668019) asin Treatment of Hek293 cells with different siRNA to quantify the amountof BDNF mRNA, using the in a same volume of Opti-MEM media (Gibcocat#31985-070). This was performed at room temperature for 20 min andapplied dropwise to one well of the 6 well plates with HepG2 cells.Similar mixture including water instead of the oligonucleotide solutionwas used for the mock-transfected controls.

Results:

As shown here in FIG. 1 e, there is a dose-dependent up-regulation ofBDNF when BDNF-AS is targeted by mBDNF-AntagoNAT9.

FIG. 1 shows Antisense-mediated regulation of sense mRNA and protein.(A) Knockdown of brain derived neurotrophic factor (BDNF) naturalantisense transcript, BDNF-AS, in HEK293T cells (n=12 per treatment)with each of three unique siRNAs (10 nM) targeting the non-overlappingregion of BDNF-AS transcript, caused 2-6 fold upregulation of BDNF(sense) mRNA (n=6 for each data point/treatment ***=P<0.001, **=P<0.01).Similar results were obtained from experiments using Human corticalneuron (HCN), glioblastoma (MK059) cells, mouse N2a cells andneurospheres “data not shown”. Scrambled sequences, mock transfectionand control siRNAs were used as controls. Control siRNA for this andother experiments is an inert siRNA (CCUCUCCACGCGCAGUACATT) that doesnot target any known sequence in the mammalian genome. All measurementswere normalized to the 18S rRNA and graphed as a percentage of each mRNAto the negative siRNA control sample.

(B) Changes in BDNF and BDNF-AS transcripts were assessed over a periodof time, following BDNF-AS knockdown (n=6 for each datapoint/treatment). siRNA knockdown of human BDNF-AS resulted in efficientand consistent downregulation of BDNF-AS, starting at 6 h and continuingon to 72 h. BDNF mRNA levels rose at 18 h, remaining high for more than72 h, reversing to pre-treatment levels at 96 h. Note that the peak at48 h is consistent and reproducible. Although BDNF-AS knockdown beginsafter 6 h, upregulation of BDNF started 18 h post-treatment. This timelag between the depletion of BDNF-AS and the increase of BDNF mRNA showsthe sequential order of events indicating that the cells require time toadapt to the removal of the antisense transcript before upregulatingBDNF.

(C) siRNA-mediated knockdown of BDNF-AS transcript caused an increase inBDNF protein levels measured by ELISA. Cells were transfected with 10 nMof two active siRNAs for BDNF-AS, scrambled siRNAs or a control siRNAfor 48 hours. The supernatants of these cells were concentrated andanalyzed for BDNF protein by ELISA, using a commercially available kit.BDNF protein was significantly increased (n=6 per treatment,***=P<0.0001, **=P<0.001) with siRNA targeting BDNF-AS transcript.

(D) Western blots confirmed that knockdown of the non-protein-codingBDNF-AS, with BDNF-AS siRNA1, but not control non-targeting siRNAtranscript increased BDNF protein levels without changing the levels ofbeta-actin. Collectively, these data suggest that there is a discordantrelationship between the sense and antisense BDNF transcripts in whichBDNF-AS suppresses the expression of BDNF mRNA and protein. Removal ofthis negative regulatory effect, by BDNF-AS knockdown, causesupregulation of BDNF mRNA and protein levels.

(E) Dose-dependent increases in Bdnf following Bdnf-AS depletion: doseresponse experiments were performed using 11 different concentrations(1:3 serial dilutions ranging from 300 nM to 5 pM) of mBdnf-AntagoNAT9(n=6 per data point/treatment) and a dose-dependent increase wasobserved in Bdnf mRNA levels at 1-300 nM concentration with an EC50 of6.6 nM.

Treatment of Hippocampal Neurospheres with siRNA

Dissecting Mouse Hippocampal Neural Stem Cells in Neurospheres:

neuronal stem cells were separated from the hippocampus of mouse pups,P0-P1. The hippocampi were mechanically separated to single cells,collected by short spins and grown in a mixture of DMEM and F12,containing glutamine, antibiotics, B27 solution and 0.001 mMconcentration of both EGF and FGF. After 3-4 days floating neurospheresformed. 100,000 cells were plated in 24-well plates coated withpoly-L-Lysine (PLL). The plating of neurosphere cells onto PLL willstart the differentiation process. On the third day post-plating, growthfactors were removed from the medium and allowed the cells to grow for 4more days (7 days post-plating). By this time, the cell culture had amix of neural cell lineages consisting of astrocytes, neurons,oligodendrocytes and their progenitors making it more similar to maturebrain tissue. The expression of Bdnf and Bdnf-AS was measured infloating neurospheres as well as in 3 and 7 days post-plating cultures.Knockdown experiments were performed, using either 50 nM siRNAs or 20 nMantisense oligonucleotides targeting Bdnf-AS transcript, at 3 or 7 dayspost-plating. Neural stem cells are also seeded in immunocytochemistrychambers, (18,000 cell per well) in a total volume of 80 μl.Neurospheres were then transfected, using the same protocol, to assessthe functional effects of Bdnf-AS knockdown on murine primary cells.After 48-72 h, cells were fixed with paraformaldehyde (4%) for 20 minand washed with 1×PBS several times. After blocking with FBS,neurospheres were incubated with primary antibody (Monoclonal Rabbit βtubulin III, TUJ1) at a 1:2000 concentration overnight. Fixed cells wereincubated with secondary antibody, labeled with Alexafluor 568 (goatanti-rabbit IgG, 2 mg/ml, at concentration of 1:5000). Nuclei werestained with Hoechst stain. Images were obtained by immunofluorescenceantigen detection microscopy.

Targeting of BDNF-AS by AntagoNATs:

The term AntagoNAT is used here to describe single-strandedoligonucleotide molecules that inhibit sense-antisense interactions(with different modifications, see supplementary methods).Single-stranded gapmer were designed, oligonucleotides, 14 nucleotidesin length, with 2′O-Methyl RNA and/or locked nucleic acid (LNA)modifications. Using this strategy, we tiled the entire overlappingregion between human BDNF-AS and BDNF transcripts and identified severalefficacious AntagoNATs capable of upregulating of BDNF mRNA.hBDNF-AntagoNAT1 and hBDNF-AntagoNAT4, targeting the first part of theoverlapping region, produced the largest response. The data suggeststhat blockage of BDNF antisense RNA, by single-stranded AntagoNATs, issufficient in causing an increase in BDNF mRNA.

Then single-stranded gapmer were designed, LNA-modified, 15 DNAoligonucleotides (AntagoNATs) 16-nucleotides in length withphosphorothioate backbone, complementary to mouse Bdnf-AS. TwoAntagoNATs (mBdnf-AntagoNAT3 and mBdnf-AntagoNAT9) consistently showed astatistically significant increase in Bdnf mRNA levels in mouse N2acells (FIG. 7).

FIG. 7 shows Inhibition of the human BDNF-AS transcript byhBDNFAntagoNAT: The BDNF-AS transcript contains a 225-nucleotideoverlapping region that has full complementarity to the BDNF mRNA.RNA-RNA interactions may be responsible for the discordant regulation ofBDNF by its antisense transcript. To determine the regulatory role ofBDNF-AS on BDNF mRNA, gapmers (AntagoNATs) containing both LNA and 2′OMeRNA modification were utilized to block the interaction between senseand antisense transcripts. overlapping region was covered by tilinghBDNF-AatagoNATs. It was found that the use of hBDNF-AntagoNATsupregulates the BDNF mRNA. Marginal downregulation of BDNF-AS transcriptwas observed, which was not expected for 2′OMe-RNA containing blockingoligos. 16 hBDNF-AntagoNATs (14-mers each with the sequences providedbelow) were tested and it was found that blocking the first half of theBDNF-AS overlapping region has a greater effect on the upregulation ofBDNF mRNA. Specifically, hBDNF-AntagoNAT1 and hBDNFAntagoNAT4 causedsignificant upregulation of BDNF mRNA. Unlike synthetic siRNAs,antisense oligonucleotides are single-stranded and can be shorter inlength; therefore, reducing non-specific (off-target) binding effects.Single-stranded locked nucleic acid (LNA)-modified oligonucleotides aregenerally more effective, in vivo, compared to unmodified siRNAs.Bdnfupregulation increases neuronal outgrowth.

Bdnf Upregulation Increases Neuronal Outgrowth:

Consistent with many previous reports that indicate stimulatory effectsof Bdnf on neuronal outgrowth and adult neurogenesis16-17, it was foundthat an increase in the endogenous Bdnf level due to the knockdown ofBdnf-AS transcript resulted in increased neuronal cell number and inneurite outgrowth and maturation at 3 and 7 days post-plating inneurospheres (FIG. 5 a-d). These data suggest that the upregulation ofendogenous Bdnf, due to inhibition of antisense RNA, induces neuronaldifferentiation in neuronal progenitor cells and might cause a maturephenotype in nascent neurons.

Results:

FIG. 2 shows Bdnf upregulation increases neuronal outgrowth (A-B)Immunocytochemistry images of hippocampal neurospheres treated witheither control siRNA (A) or Bdnf-AS siRNA (B) 3 d post-platting. (C-D)Immunocytochemistry images of neuronal maturation and neurite outgrowthin hippocampal neurospheres treated with either control siRNA (C) orBdnf-AS siRNA (D) 7 d post-plating. Treatment of cells with siRNAtargeting the Bdnf-AS transcript resulted in increased neuronal cellnumber as well as increase in neurite outgrowth and maturation, both at3 d or 7 d postplating neurospheres. B-tubulin III stained red, GFAPstained green and DAPI stained blue.

Delivery Intracerebroventicular (ICV) of mBDNF-AntagoNAT9 Using OsmoticMini-Pumps Knockdown BDNF-AS and Up-Regulate BDNF

Mouse Studies:

10 eight-week-old male C57BL/6 mice were used for in vivo experiments.The mice were prepared with chronic indwelling cannulae in the dorsalthird ventricle implanted subcutaneously with osmotic mini-pumps thatdelivered continuous infusions (0.11 microliter/h) of syntheticantisense oligonucleotide directed against Bdnf-AS (mBdnf-AntagoNAT9) orcontrol oligonucleotide (inert sequence that does not exist in human ormouse) at a dose of 1.5 mg/kg/d for 4 weeks. Tubing was connected to theexit port of the osmotic minipump and tunneled subcutaneously to theindwelling cannula, such that the treatments were delivered directlyinto the brain. At 5 d post-implantation all animals received dailyintra-peritoneal (IP) injection of BrdU (80 mg/kg), for five consecutivedays. At the 28th day post-surgery, the animals were sacrificed andthree tissues were excised (hippocampus, frontal cortex and cerebellum)from each mouse brain for quantitative RNA measurements.

Knockdown of Bdnf-AS Increases Bdnf In Vivo:

Osmotic mini-pumps for intracerebroventricular (ICV) delivery ofmBdnf-AntagoNAT9 to C57BL/6 mice were utilized. mBdnf-AntagoNAT9 werethen selected, which is targeting a non-overlapping region of mouseBdnf-AS, over other active AntagoNATs, based on its high efficacy toincrease in Bdnf mRNA in vitro. After 28 days of continuous AntagoNATinfusion, Bdnf mRNA levels were increased across forebrain regionsadjacent to the third ventricle in mice treated with mBdnf-AntagoNAT9 ascompared to levels unaltered by an inert control oligonucleotide (FIG. 3a,b). Bdnf and Bdnf-AS transcripts were unaltered in the hypothalamus, astructure that is not immediately adjacent to the third ventricle (FIG.3 c). Moreover, it was found that AntagoNAT-mediated blockade of Bdnf-ASresults increased Bdnf protein levels (FIG. 3 d,e). These findingscorrespond with the in vitro data described above and indicate that theblockade of Bdnf-AS results in the increase of Bdnf mRNA and proteinexpression in vivo.

RNA Extraction and RT-PCR of the Mouse Brain Samples:

Mice were euthanized after 28 days and the brains were excised. Onehemibrain from each mouse was fixed in 4% formaldehyde overnight forhistological studies. Another hemibrain was excised for RNA quantitativemeasurement from the hippocampus, frontal cortex and cerebellum. RNA wasextracted after homogenization in Trizol reagent (Invitrogen, 15596-026)according to the manufacturer's protocol. The aqueous phase wasseparated and added an equal volume of 70% ethanol before passing thesamples through Qiagen RNeasy columns (QIAGEN, 74106) and those RNAsamples were subjected to on-column DNAse treatment for removal of DNAcontamination. 400 ng of each sample was used for the first strand cDNAsynthesis and RT-PCR measurements were carried out. The percentilechanges were plotted in RNA levels, for individual tissues as comparedto control mice, in each graph.

Results:

FIG. 3 shows Bdnf-AS regulates Bdnf mRNA and protein in vivo; (A-C)Using osmotic mini-pumps, mBdnf-AntagoNAT9 (CAACATATCAGGAGCC) or controloligonucleotide (CCACGCGCAGTACATG) was infused constantly over a periodof 28 d, into the third ventricle of mouse brain (n=5 per treatmentgroup *=P<0.05, **=P<0.01, ***=P<0.001). mBdnf-AntagoNAT9 directedagainst Bdnf-AS but not the control oligonucleotide resulted in anincrease in Bdnf levels in the hippocampus (A) and frontal cortex (B).In the hypothalamus (C) both transcripts were unchanged, as was expectedfor a tissue that is not directly connected to the third ventricle ofthe brain. (D-E) BDNF protein levels were assessed by ELISA and foundthat mBdnf-AntagoNAT9 treatment results in an increase in BDNF protein,both in the hippocampus (D) and frontal cortex (E), as compared tocontrol oligonucleotide treated mice.

Delivery Intracerebroventicular (ICV) of mBDNF-AntagoNAT9 Using OsmoticMini-Pumps Knockdown BDNF-AS and Up-Regulate BDNF

BrdU was injected in the mice treated with mBdnf-AntagoNAT9 in the firstweek of the study for 5 days. After 28 days of continuous AntagoNATinfusion, histological examination of brain tissues was performed andneuronal proliferation was quantified and survival using Ki67 and BrdUmarkers, respectively. In mice treated with mBdnf-AntagoNAT9, anincrease was observed in Ki67 positive (proliferating) cells as comparedto control treated mice (FIG. 4 a,b). the number of Ki67 positive cellswas quantified and a significant increase in cell proliferation wasfound in mice treated with mBdnf-AntagoNAT9 compared to controloligonucleotide (FIG. 4 c). In mice treated with mBdnf-AntagoNAT9, therewas a significant increase in BrdU incorporation (surviving cells) ascompared to the control oligonucleotide-treated mice (FIG. 4 d). Therewere no differences in hippocampal volume between control andmBdnf-AntagoNAT9 treated mice (FIG. 4 e). These findings demonstratethat Bdnf-AS regulates Bdnf levels in vivo.

Results:

FIG. 4 shows Blocking of Bdnf-AS, in vivo, causes an increase inneuronal survival and proliferation; (A-B) mice were treated withmBdnf-AntagoNAT9 or control oligos. After 28 d of continuousmBdnf-AntagoNAT9 infusion, histological examination of brain tissues wasperformed, using Ki67. Ki67 is the marker of proliferating cells inhippocampus and an increase in the number of proliferating cells wasobserved in mice received Bdnf-AntagoNAT treatment compare to micereceived control oligos. In mice treated with mBdnf-AntagoNAT9 (B),there was an increase in Ki67 positive cells (proliferating cells), ascompared to control treated mice (A). (C) Mice treated withmBdnf-AntagoNAT9 had a significant increase in the number of Ki67positive cells as compared to control treated mice. (D) In mice treatedwith mBdnf-AntagoNAT9, there was a significant increase in the number ofsurviving cells (BrdU positive) as compared to control oligonucleotidetreated mice. (E) There were no differences in hippocampal volumebetween control and mBdnf-AntagoNAT9 treated mice. Together these data(n=5 per treatment group *=P<0.05, ***=P<0.001) demonstrates thatBdnf-AS regulates Bdnf levels in vivo and that blocking Bdnfsense-antisense interactions results in an increase in neuronal lineage,proliferation and survival.

Although the invention has been illustrated and described with respectto one or more implementations, equivalent alterations and modificationswill occur to others skilled in the art upon the reading andunderstanding of this specification and the annexed drawings. Inaddition, while a particular feature of the invention may have beendisclosed with respect to only one of several implementations, suchfeature may be combined with one or more other features of the otherimplementations as may be desired and advantageous for any given orparticular application.

The Abstract of the disclosure will allow the reader to quicklyascertain the nature of the technical disclosure. It is submitted withthe understanding that it will not be used to interpret or limit thescope or meaning of the following claims.

What is claimed is:
 1. A method of modulating a function of and/or theexpression of a Brain derived neurotrophic factor (BDNF) polynucleotidein a biological system comprising: contacting said system with at leastone antisense oligonucleotide 5 to 30 nucleotides in length wherein saidat least one oligonucleotide has at least 50% sequence identity to areverse complement of a natural antisense of a Brain derivedneurotrophic factor (BDNF) polynucleotide; thereby modulating a functionof and/or the expression of the Brain derived neurotrophic factor (BDNF)polynucleotide, with the proviso that the oligonucleotides having SEQ IDNOS 50-55 are excluded.
 2. A method of modulating a function of and/orthe expression of a Brain derived neurotrophic factor (BDNF)polynucleotide in a biological system according to claim 1 comprising:contacting said biological system with at least one antisenseoligonucleotide 5 to 30 nucleotides in length wherein said at least oneoligonucleotide has at least 50% sequence identity to a reversecomplement of a polynucleotide comprising 5 to 30 consecutivenucleotides within the natural antisense transcript nucleotides 1 to1279 of SEQ ID NO: 3 or 1 to 1478 of SEQ TD NO: 4 or 1 to 1437 of SEQ IDNO: 5 or 1 to 2322 of SEQ ID NO: 6 or 1 to 2036 of SEQ ID NO: 7 or 1 to2364 of SEQ ID NO: 8 or 1 to 3136 of SEQ ID NO: 9 or 1 to 906 of SEQ IDNO: 10 or 1 to 992 of SEQ ID NO: 11, with the proviso that theoligonucleotides having SEQ ID NOS 50-55 are excluded. therebymodulating a function of and/or the expression of the Brain derivedneurotrophic factor (BDNF) polynucleotide.
 3. A method of modulating afunction of and/or the expression of a Brain derived neurotrophic factor(BDNF) polynucleotide in patient cells or tissues in vivo or in vitrocomprising: contacting said cells or tissues with at least one antisenseoligonucleotide 5 to 30 nucleotides in length wherein saidoligonucleotide has at least 50% sequence identity to an antisenseoligonucleotide to the Brain derived neurotrophic factor (BDNF)polynucleotide; thereby modulating a function of and/or the expressionof the Brain derived neurotrophic factor (BDNF) polynucleotide inpatient cells or tissues in vivo or in vitro, with the proviso that theoligonucleotides having SEQ ID NOS 50-55 are excluded.
 4. A method ofmodulating a function of and/or the expression of a Brain derivedneurotrophic factor (BDNF) polynucleotide in patient cells or tissuesaccording to claim 3 comprising: contacting said biological system withat least one antisense oligonucleotide 5 to 30 nucleotides in lengthwherein said at least one oligonucleotide has at least 50% sequenceidentity to a reverse complement of a polynucleotide comprising 5 to 30consecutive nucleotides within the natural antisense transcriptnucleotides 1 to 1279 of SEQ ID NO: 3 or 1 to 1478 of SEQ ID NO: 4 or 1to 1437 of SEQ ID NO: 5 or 1 to 2322 of SEQ ID NO: 6 or 1 to 2036 of SEQID NO: 7 or 1 to 2364 of SEQ ID NO: 8 or 1 to 3136 of SEQ ID NO: 9 or 1to 906 of SEQ ID NO: 10 or 1 to 992 of SEQ ID NO: 11, with the provisothat the oligonucleotides having SEQ ID NOS 50-55 are excluded, therebymodulating a function of and/or the expression of the Brain derivedneurotrophic factor (BDNF) polynucleotide.
 5. A method of modulating afunction of and/or the expression of a Brain derived neurotrophic factor(BDNF) polynucleotide in a biological system comprising: contacting saidsystem with at least one antisense oligonucleotide that targets a regionof a natural antisense oligonucleotide of the Brain derived neurotrophicfactor (BDNF) polynucleotide; thereby modulating a function of and/orthe expression of the Brain derived neurotrophic factor (BDNF)polynucleotide, with the proviso that the oligonucleotides having SEQ IDNOS 50-55 are excluded.
 6. The method of claim 5, wherein a function ofand/or the expression of the Brain derived neurotrophic factor (BDNF) isincreased in vivo or in vitro with respect to a control.
 7. The methodof claim 5, wherein the at least one antisense oligonucleotide targets anatural antisense sequence of a Brain derived neurotrophic factor (BDNF)polynucleotide.
 8. The method of claim 5, wherein the at least oneantisense oligonucleotide targets a nucleic acid sequence comprisingcoding and/or non-coding nucleic acid sequences of a Brain derivedneurotrophic factor (BDNF) polynucleotide.
 9. The method of claim 5,wherein the at least one antisense oligonucleotide targets overlappingand/or non-overlapping sequences of a Brain derived neurotrophic factor(BDNF) polynucleotide.
 10. The method of claim 5, wherein the at leastone antisense oligonucleotide comprises one or more modificationsselected from: at least one modified sugar moiety, at least one modifiedinternucleoside linkage, at least one modified nucleotide, andcombinations thereof.
 11. The method of claim 10, wherein the one ormore modifications comprise at least one modified sugar moiety selectedfrom: a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modifiedsugar moiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugarmoiety, and combinations thereof.
 12. The method of claim 10, whereinthe one or more modifications comprise at least one modifiedinternucleoside linkage selected from: a phosphorothioate,2′-Omethoxyethyl (MOE), 2′-fluoro, alkylphosphonate, phosphorodithioate,alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphatetriester, acetamidate, carboxymethyl ester, and combinations thereof.13. The method of claim 10, wherein the one or more modificationscomprise at least one modified nucleotide selected from: a peptidenucleic acid (PNA), a locked nucleic acid (LNA), an arabino-nucleic acid(FANA), an analogue, a derivative, and combinations thereof.
 14. Themethod of claim 1, wherein the at least one oligonucleotide comprises atleast one oligonucleotide sequences set forth as SEQ ID NOS: 12 to 49.15. A method of modulating a function of and/or the expression of aBrain derived neurotrophic factor (BDNF) gene in mammalian cells ortissues in vivo or in vitro comprising: contacting said cells or tissueswith at least one short interfering RNA (siRNA) oligonucleotide 5 to 30nucleotides in length, said at least one siRNA oligonucleotide beingspecific for an antisense polynucleotide of a Brain derived neurotrophicfactor (BDNF) polynucleotide, wherein said at least one siRNAoligonucleotide has at least 50% sequence identity to a complementarysequence of at least about five consecutive nucleic acids of theantisense and/or sense nucleic acid molecule of the Brain derivedneurotrophic factor (BDNF) polynucleotide; and, modulating a function ofand/or the expression of Brain derived neurotrophic factor (BDNF) inmammalian cells or tissues in vivo or in vitro.
 16. The method of claim15, wherein said oligonucleotide has at least 80% sequence identity to asequence of at least about five consecutive nucleic acids that iscomplementary to the antisense and/or sense nucleic acid molecule of theBrain derived neurotrophic factor (BDNF) polynucleotide.
 17. Anoligonucleotide 5 to 30 nucleotides in length wherein saidoligonucleotide has at least 50% sequence identity to a reversecomplement of a polynucleotide comprising 5 to 30 consecutivenucleotides within the natural antisense transcript nucleotides 1 to1279 of SEQ ID NO: 3 or 1 to 1478 of SEQ ID NO: 4 or 1 to 1437 of SEQ IDNO: 5 or 1 to 2322 of SEQ ID NO: 6 or 1 to 2036 of SEQ ID NO: 7 or 1 to2364 of SEQ ID NO: 8 or 1 to 3136 of SEQ ID NO: 9 or 1 to 906 of SEQ IDNO: 10 or 1 to 992 of SEQ ID NO: 11, with the proviso that theoligonucleotides having SEQ ID NOS 50-55 are excluded, and optionallyfurther comprising at least one modification wherein the at least onemodification is selected from: at least one modified sugar moiety; atleast one modified internucleotide linkage; at least one modifiednucleotide, and combinations thereof; wherein said oligonucleotide is anantisense compound which hybridizes to and modulates the function and/orexpression of a Brain derived neurotrophic factor (BDNF) gene in vivo orin vitro as compared to a normal control.
 18. The oligonucleotideaccording to claim 17 wherein said oligonucleotide is 5 to 30nucleotides in length and has at least 50% sequence identity to thereverse complement of 5-30 consecutive nucleotides within a naturalantisense transcript of the BDNF gene.
 19. The oligonucleotide of claim18, wherein the at least one modification comprises an internucleotidelinkage selected from the group consisting of: phosphorothioate,alkylphosphonate, phosphorodithioate, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate,carboxymethyl ester, and combinations thereof.
 20. The oligonucleotideof claim 18, wherein said oligonucleotide comprises at least onephosphorothioate internucleotide linkage.
 21. The oligonucleotide ofclaim 18, wherein said oligonucleotide comprises a backbone ofphosphorothioate internucleotide linkages.
 22. The oligonucleotide ofclaim 18, wherein the oligonucleotide comprises at least one modifiednucleotide, said modified nucleotide selected from: a peptide nucleicacid, a locked nucleic acid (LNA), analogue, derivative, and acombination thereof.
 23. The oligonucleotide of claim 18, wherein theoligonucleotide comprises a plurality of modifications, wherein saidmodifications comprise modified nucleotides selected from:phosphorothioate, alkylphosphonate, phosphorodithioate,alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphatetriester, acetamidate, carboxymethyl ester, and a combination thereof.24. The oligonucleotide of claim 18, wherein the oligonucleotidecomprises a plurality of modifications, wherein said modificationscomprise modified nucleotides selected from: peptide nucleic acids,locked nucleic acids (LNA), analogues, derivatives, and a combinationthereof.
 25. The oligonucleotide of claim 18, wherein theoligonucleotide comprises at least one modified sugar moiety selectedfrom: a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modifiedsugar moiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugarmoiety, and a combination thereof.
 26. The oligonucleotide of claim 18,wherein the oligonucleotide comprises a plurality of modifications,wherein said modifications comprise modified sugar moieties selectedfrom: a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modifiedsugar moiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugarmoiety, and a combination thereof.
 27. The oligonucleotide of claim 18,wherein the oligonucleotide is of at least about 5 to 30 nucleotides inlength and hybridizes to an antisense and/or sense strand of a Brainderived neurotrophic factor (BDNF) polynucleotide wherein saidoligonucleotide has at least about 60% sequence identity to acomplementary sequence of at least about five consecutive nucleic acidsof the antisense and/or sense coding and/or noncoding nucleic acidsequences of the Brain derived neurotrophic factor (BDNF)polynucleotide.
 28. The oligonucleotide of claim 18, wherein theoligonucleotide has at least about 80% sequence identity to acomplementary sequence of at least about five consecutive nucleic acidsof the antisense and/or sense coding and/or noncoding nucleic acidsequence of the Brain derived neurotrophic factor (BDNF) polynucleotide.29. The oligonucleotide of claim 18, wherein said oligonucleotidehybridizes to and modulates expression and/or function of at least oneBrain derived neurotrophic factor (BDNF) polynucleotide in vivo or invitro, as compared to a normal control.
 30. The oligonucleotide of claim18, wherein the oligonucleotide comprises the sequences set forth as SEQID NOS: 12 to
 49. 31. A pharmaceutical composition comprising one ormore oligonucleotides specific for one or more Brain derivedneurotrophic factor (BDNF) polynucleotides according to claim 17 and apharmaceutically acceptable excipient.
 32. The composition of claim 31,wherein the oligonucleotides have at least about 40% sequence identityas compared to any one of the nucleotide sequences set forth as SEQ IDNOS: 12 to
 49. 33. The composition of claim 31, wherein theoligonucleotides comprise nucleotide sequences set forth as SEQ ID NOS:12 to
 49. 34. The composition of claim 33, wherein the oligonucleotidesset forth as SEQ ID NOS: 12 to 49 comprise one or more modifications orsubstitutions.
 35. The composition of claim 34, wherein the one or moremodifications are selected from: phosphorothioate, methylphosphonate,peptide nucleic acid, locked nucleic acid (LNA) molecules, andcombinations thereof.
 36. A method of preventing or treating a diseaseassociated with at least one Brain derived neurotrophic factor (BDNF)polynucleotide and/or at least one encoded product thereof, comprising:administering to a patient a therapeutically effective dose of at leastone antisense oligonucleotide 5 to 30 nucleotides in length wherein saidat least one oligonucleotide has at least 50% sequence identity to areverse complement of a polynucleotide comprising 5 to 30 consecutivenucleotides within the natural antisense transcript nucleotides 1 to1279 of SEQ ID NO: 3 or 1 to 1478 of SEQ ID NO: 4 or 1 to 1437 of SEQ IDNO: 5 or 1 to 2322 of SEQ ID NO: 6 or 1 to 2036 of SEQ ID NO: 7 or 1 to2364 of SEQ ID NO: 8 or 1 to 3136 of SEQ ID NO: 9 or 1 to 906 of SEQ IDNO: 10 or 1 to 992 of SEQ ID NO: 11, with the proviso that theoligonucleotides having SEQ ID NOS 50-55 are excluded and that binds toa natural antisense sequence of said at least one Brain derivedneurotrophic factor (BDNF) polynucleotide and modulates expression ofsaid at least one Brain derived neurotrophic factor (BDNF)polynucleotide; thereby preventing or treating the disease associatedwith the at least one Brain derived neurotrophic factor (BDNF)polynucleotide and/or at least one encoded product thereof.
 37. Themethod of claim 36, wherein a disease associated with the at least oneBrain derived neurotrophic factor (BDNF) polynucleotide is selectedfrom: a disease or disorder associated with abnormal function and/orexpression of BDNF, a neurological disease or disorder, a disease or adisorder associated with defective neurogenesis; a neurodegenerativedisease or disorder (e.g., Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis etc.); aneuropsychiatric disorder (depression, schizophrenia, schizofreniformdisorder, schizoaffective disorder, and delusional disorder; anxietydisorders such as panic disorder, phobias (including agoraphobia), anobsessive-compulsive disorder, a posttraumatic stress disorder, abipolar disorder, anorexia nervosa, bulimia nervosa), an autoimmunedisorder (e.g., multiple sclerosis) of the central nervous system,memory loss, a long term or a short term memory disorder, benignforgetfulness, a childhood learning disorder, close head injury, anattention deficit disorder, neuronal reaction to viral infection, braindamage, narcolepsy, a sleep disorder (e.g., circadian rhythm disorders,insomnia and narcolepsy); severance of nerves or nerve damage, severanceof cerebrospinal nerve cord (CNS) and a damage to brain or nerve cells,a neurological deficit associated with AIDS, a motor and tic disordercharacterized by motor and/or vocal tics (e.g., Tourette's disorder,chronic motor or vocal tic disorder, transient tic disorder, andstereotypic movement disorder), a substance abuse disorder (e.g.,substance dependence, substance abuse and the sequalae of substanceabuse/dependence, such as substance-induced psychological disorder,substance withdrawal and substance-induced dementia or amnesticdisorder), traumatic brain injury, tinnitus, neuralgia (e.g., trigeminalneuralgia) pain (e.g., chronic pain, chronic inflammatory pain, painassociated with arthritis, fibromyalgia, back pain, cancer-associatedpain, pain associated with digestive disease, pain associated withCrohn's disease, pain associated with autoimmune disease, painassociated with endocrine disease, pain associated with diabeticneuropathy, phantom limb pain, spontaneous pain, chronic post-surgicalpain, chronic temporomandibular pain, causalgia, post-herpeticneuralgia, AIDS-related pain, complex regional pain syndromes type I andII, trigeminal neuralgia, chronic back pain, pain associated with spinalcord injury, pain associated with drug intake and recurrent acute pain,neuropathic pain), inappropriate neuronal activity resulting inneurodysthesias in a disease such as diabetes, an MS and a motor neurondisease, ataxias, muscular rigidity (spasticity), temporomandibularjoint dysfunction, Reward deficiency syndrome (RDS), neurotoxicitycaused by alcohol or substance abuse (e.g., ecstasy, methamphetamineetc.), mental retardation or cognitive impairment (e.g., nonsyndromicX-linked mental retardation, fragile X syndrome, Down's syndrome,autism), aphasia, Bell's palsy, Creutzfeldt-jacob disease, encephalitis,age related macular degeneration, ondine syndrome, WAGR syndrome,hearing loss, Rett syndrome, epilepsy, spinal cord injury, stroke,hypoxia, ischemia, brain injury, diabetic neuropathy, peripheralneuropathy, nerve transplantation complications, motor neuron disease,peripheral nerve injury, obesity, a metabolic syndrome, cancer, asthma,an atopic disease, inflammation, allergy, eczema, a neuro-oncologicaldisease or disorder, neuro-immunological disease or disorder andneuro-otological disease or disorder; and a disease or disorderassociated with aging and senescence.
 38. Use of an oligonucleotideselected from the group consisting of SEQ ID NOS 50-55 to target anatural antisense transcript (“NAT”) of a BDNF polynucleotide tomodulate the expression of the BDNF polynucleotide wherein said NATs areselected from the group consisting of SEQ ID NOS. 3 to
 11. 39. Use of anoligonucleotide selected from the group consisting of SEQ ID NOS 50-55to target a natural antisense transcript (NAT) of a BDNF polynucleotideto modulate the expression of the BDNF polynucleotide wherein said NATis selected from the group consisting of SEQ ID NOS. 3, 4, 5, 7, 8, 9,10 and 11.